CN115397848A - TAT peptide binding proteins and uses thereof - Google Patents

TAT peptide binding proteins and uses thereof Download PDF

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CN115397848A
CN115397848A CN202180025971.XA CN202180025971A CN115397848A CN 115397848 A CN115397848 A CN 115397848A CN 202180025971 A CN202180025971 A CN 202180025971A CN 115397848 A CN115397848 A CN 115397848A
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tat
seq
domain
amino acid
acid sequence
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徐欣
E·J·瓦格纳
B·鲁普
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United States Represented By Secretary Of Health And Human Services
Lalima Biomedical Co
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United States Represented By Secretary Of Health And Human Services
Lalima Biomedical Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1072—Regulatory proteins, e.g. tat, rev, vpt
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/163—Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Abstract

The present invention includes TAT peptide binding proteins. In particular, the invention relates to antibodies that specifically bind to a TAT protein transduction domain. The antibodies of the invention may be full-length antibodies or antigen-binding portions thereof. Also provided herein are methods of making the antibodies of the invention and methods of detecting, quantifying, purifying, and isolating TAT protein transduction domains (e.g., TAT fusion molecules comprising TAT protein transduction domains) using the antibodies of the invention, as well as methods of diagnosing and monitoring HIV and AIDS.

Description

TAT peptide binding proteins and uses thereof
RELATED APPLICATIONS
This application claims priority from U.S. provisional patent application No. 62/970,662, filed on 5/2/2020, which is incorporated herein by reference in its entirety.
Sequence listing
The present application contains a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy was created on 2.5.2021, named 130197-00220_SL.txt and was 27400 bytes in size.
Background
The human immunodeficiency virus type 1 (HIV-1) TAT protein is a key regulatory protein in the replication cycle of HIV-1. The wild-type TAT gene of HIV-1 is essential for viral RNA production and viral replication. TAT interacts with cellular transcription factors and cytokines, such as tumor necrosis factor-alpha (TNF-a), and alters the expression of various genes in HIV-1 infected and uninfected cells.
TAT has also been shown to be taken up and internalized by cells. Thus, fusion of heterologous proteins to TAT has been used as a means of cell delivery of heterologous proteins in cell culture and in living animals.
The presence of TAT-specific cytotoxic T lymphocytes is associated with strong resistance to HIV infection (Allen et al, nature 2000 (6802): 386 390). TAT-mediated pathogenic effects can also be neutralized by anti-TAT antibodies. Antibodies directed against TAT conserved regions, such as cysteine-rich and lysine-rich domains, have been shown to be particularly effective in inhibiting HIV replication. In HIV-1 infected patients, a strong humoral immune response to the HIV-1TAT protein is negatively correlated with peripheral blood disease virulence load (Re et al, J.Clin.Virol.2001.21 (1): 819).
There remains a need in the art for anti-TAT antibodies that can be used to detect and quantify TAT peptides, such as TAT peptides used as part of fusion molecules for cellular delivery of heterologous cargo (cargo) moieties. There is also a need for anti-TAT antibodies for therapeutic purposes in the treatment of HIV infection.
Disclosure of Invention
In certain aspects, the invention provides anti-TAT binding proteins, e.g., antibodies and antigen-binding portions thereof, that bind to a TAT protein transduction domain.
In one aspect, the invention provides a binding protein comprising an antigen binding domain comprising a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID No. 4, wherein the binding protein is capable of binding to a TAT protein transduction domain.
In some embodiments, the antigen binding domain further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3.
In some embodiments, the antigen binding domain further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2.
In some embodiments, the antigen binding domain further comprises a light chain CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID No. 8 and SEQ ID No. 12.
In some embodiments, the antigen binding domain further comprises a light chain CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID No. 7 and SEQ ID No. 11.
In some embodiments, the antigen binding domain further comprises a light chain CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 6 and SEQ ID NO 10.
In another aspect, the present invention provides a binding protein comprising an antigen binding domain comprising: a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 3, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 2; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 7, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 6; or a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 12, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 11, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 10, wherein the binding protein is capable of binding to a TAT protein transduction domain.
In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 1 or SEQ ID NO 14.
In some embodiments, the antigen binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 5, a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO.9, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 13.
In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 5.
In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 9.
In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 13.
In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 14 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 5.
In another aspect, the invention provides a binding protein comprising an antigen binding domain comprising a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID No. 18, wherein the binding protein is capable of binding to a TAT protein transduction domain.
In some embodiments, the antigen binding domain further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO 17.
In some embodiments, the antigen binding domain further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID No. 16.
In some embodiments, the antigen binding domain further comprises a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO. 22.
In some embodiments, the antigen binding domain further comprises a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO. 21.
In some embodiments, the antigen binding domain further comprises a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO. 20.
In another aspect, the invention provides a binding protein comprising an antigen binding domain comprising: a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 18, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 17, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 16; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:22, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:21, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:20, wherein the binding protein is capable of binding to a TAT protein transduction domain.
In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 15.
In some embodiments, the antigen binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO 19.
In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 15 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 19.
In some embodiments, the TAT protein transduction domain comprises the amino acid sequence of SEQ ID No. 23.
In some embodiments, the TAT protein transduction domain consists essentially of the amino acid sequence of SEQ ID No. 23.
In some embodiments, the TAT protein transduction domain is covalently linked to a cargo moiety.
In some embodiments, the cargo moiety is a polypeptide.
In some embodiments, the cargo moiety is an ataxin (frataxin) polypeptide.
In some embodiments, the cargo moiety is an antibody.
In some embodiments, the cargo moiety is a nucleic acid. In some embodiments, the nucleic acid is an siRNA, shRNA, miRNA, phosphorothioate modified RNA, aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof.
In some embodiments, the cargo moiety is a small molecule, a liposome-encapsulated protein, a radionuclide or a radionuclide-labeled compound, or any combination thereof.
In some embodiments, the binding protein is capable of binding to a TAT protein transduction domain covalently linked to a cargo moiety.
In some embodiments, the antigen binding domain binds to an epitope comprising amino acid residues of SEQ ID No. 23.
In some embodiments, the binding protein disclosed herein is an antibody.
In another aspect, the invention provides an antibody construct comprising a binding protein of the invention, further comprising a linker polypeptide or an immunoglobulin constant region.
In some embodiments, the binding protein is selected from the group consisting of: immunoglobulin molecules, monoclonal antibodies, murine antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, single domain antibodies, fv, disulfide-linked Fv, scFv, diabody, fab ', F (ab') 2 Multi-specific antibodies, dual specific antibodies (dual specific antibodies) and bispecific antibodies.
In some embodiments, the antibody construct comprises a binding protein comprising a heavy chain immunoglobulin constant region selected from the group consisting of seq id no: igM constant region, igG 4 Constant region, igG 1 Constant region, igE constant region, igG 2 Constant region, igG 3 Constant region and IgA constant region.
In some embodiments, the heavy chain immunoglobulin constant region is not an IgM.
In another aspect, the invention provides an isolated nucleic acid encoding the binding protein amino acid sequence of the invention.
In another aspect, the invention provides an isolated nucleic acid encoding an amino acid sequence of an antibody construct of the invention.
In another aspect, the invention provides a vector comprising an isolated nucleic acid of the invention.
In some embodiments, the carrier is selected from the group consisting of: pcDNA, pTT3, pEFBOS, pBV, pJV, pBJ, pGEX, VSV, pBR322, pCMV-HA, pEN, YAC, BAC, lambda phage (bacteriophamda), phagemid (Phagemid), pCAS9, pCEN6, pYES1L, p3HPRT1, pFN2A, pBC, pTZ, pGEM, pGEMK, pEX, pSAR, pCEP, cosmid (Cosmid), pBluescript, pKJK, pFloxin, pCP, pHR, pUC and pMAL.
In another aspect, the invention provides a host cell comprising a vector of the invention.
In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the prokaryotic host cell is an escherichia coli cell. In some embodiments, the eukaryotic cell is selected from the group consisting of: protist cells, insect cells, animal cells, plant cells and fungal cells. In some embodiments, the animal cell is a mammalian cell or an avian cell. In some embodiments, the host cell is selected from the group consisting of: CHO cells, COS cells, yeast cells, insect Sf9 cells, HEK-293 cells, expCHO cells, expi-293f cells, and E.coli cells. In some embodiments, the yeast cell is a saccharomyces cerevisiae cell.
In another aspect, the invention provides a method of producing an antibody, or antigen-binding portion thereof, comprising culturing a host cell in a culture medium, thereby expressing the isolated nucleic acid and producing the antibody.
In another aspect, the invention provides antibodies produced according to the methods of the invention.
In another aspect, the invention provides a transgenic mouse comprising a host cell described herein, wherein the mouse expresses a polypeptide encoded by the nucleic acid, or an antigen-binding portion thereof, that binds to a TAT protein transduction domain.
In another aspect, the invention provides a hybridoma that produces an antibody construct as described herein.
In some embodiments, the binding proteins of the invention are immobilized on a solid support. In some embodiments, the solid support is a plate, bead, or chromatography resin. In some embodiments, the bead or chromatography resin comprises protein a sepharose or protein G sepharose.
In some embodiments, the binding protein of the invention is conjugated to a detection molecule.
In some embodiments, the detection molecule is horseradish peroxidase, ruthenium terpyridyl (sulfotag), alkaline phosphatase, cresyl violet, quantum dots, FITC, an infrared molecule, a radioisotope label, or an EPR spin tracer tag.
In another aspect, the invention provides a method of detecting and/or quantifying the level of a TAT fusion molecule in a sample, comprising contacting the sample with a binding protein of the invention under conditions such that said binding protein binds to a TAT protein transduction domain in said sample, thereby detecting and/or quantifying the level of the TAT fusion molecule in the sample.
In some embodiments, the sample is a biological sample.
In some embodiments, the biological sample is a liquid sample or a tissue sample.
In some embodiments, the TAT fusion molecule comprises a TAT protein transduction domain covalently linked to a cargo moiety.
In some embodiments, the cargo moiety is a polypeptide.
In some embodiments, the cargo moiety is an ataxin polypeptide.
In some embodiments, the cargo moiety is an antibody.
In some embodiments, the cargo moiety is a nucleic acid. In some embodiments, the nucleic acid is an siRNA, shRNA, miRNA, phosphorothioate modified RNA, aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof.
In some embodiments, the cargo moiety is a small molecule, a liposome-encapsulated protein, a radionuclide or a radionuclide-labeled compound, or any combination thereof.
In some embodiments, the stability of the TAT fusion molecule is assessed.
In another aspect, the invention provides a method of isolating and/or purifying a TAT fusion molecule present in a mixture, wherein the TAT fusion molecule comprises a TAT protein transduction domain covalently linked to a cargo moiety, the method comprising (a) contacting a mixture comprising the TAT fusion molecule with an immobilized binding protein of the invention under conditions such that the TAT fusion molecule binds to the immobilized binding protein; (b) eluting the TAT fusion molecule from the immobilized binding protein.
In another aspect, the invention provides a kit comprising at least one reagent for specifically detecting the level of a TAT protein transduction domain, wherein the detection reagent is a binding protein of the invention. In some embodiments, the TAT protein transduction domain is covalently linked to a cargo moiety. In some embodiments, the kit further comprises instructions for detecting, quantifying, or characterizing the TAT protein transduction domain.
In another aspect, the invention provides a method of inhibiting translocation (translocation) of a TAT fusion molecule across a cell membrane, comprising contacting the TAT fusion molecule with an antigen binding protein of the invention, thereby inhibiting translocation of the TAT fusion molecule across a cell membrane.
In another aspect, the invention provides a method of inhibiting HIV-TAT protein activity in a subject, comprising administering to the subject an antigen binding protein of the invention, thereby inhibiting HIV-TAT protein activity in the subject.
Drawings
FIG. 1 illustrates that polyclonal antibodies raised against the entire TAT-FXN fusion molecule recognize drugs, but do not recognize them in a TAT-specific manner.
FIG. 2 illustrates that polyclonal antibodies raised against KLH-TAT do not recognize mature ataxin, but recognize BSA-TAT, which shows specificity for TAT epitopes.
FIG. 3 is a bar graph showing the relative peak areas generated by the ataxin-derived tryptase peptides following immunopurification of exemplary TAT ataxin fusion molecules using anti-TAT rabbit polyclonal antibodies (AC 1058) and commercially available anti-ataxin antibodies (ab 124680, ab113691 and ab 110328) and commercially available anti-TAT antibodies (ab 63957).
FIGS. 4A and 4B show the ability of 9 of the 10 anti-TAT antibodies of the invention to capture an exemplary TAT ataxin fusion molecule in a Pharmacokinetic (PK) assay in human plasma.
FIG. 5A shows exemplary TAT ataxin fusion molecules that bind to human plasma with 9 of the 10 anti-TAT antibodies of the invention. Figure 5B depicts the results of an anti-drug antibody (ADA) assay in human plasma, which indicates that polyclonal anti-TAT antibodies can be used as a positive control in an ADA assay.
Detailed Description
The present invention relates to TAT peptide binding proteins that bind to a TAT protein transduction domain, in particular anti-TAT peptide antibodies or antigen-binding portions thereof, including TAT fusion molecules comprising a TAT protein transduction domain, and uses thereof. Various aspects of the invention relate to antibodies and antibody fragments, conjugates thereof, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such antibodies and fragments. In one aspect, the invention relates to a binding protein comprising an antigen binding domain, wherein the binding protein is capable of binding to a TAT protein transduction domain covalently linked to a cargo moiety. In some embodiments, the cargo moiety is a polypeptide. In some embodiments, the cargo moiety is an ataxin polypeptide. In some embodiments, the cargo moiety is an antibody.
In some embodiments, the cargo moiety is a pharmacologically active compound, a small molecule, a liposome-encapsulated protein, a radionuclide or radionuclide-labeled compound, a nucleic acid (e.g., siRNA, shRNA, miRNA, phosphorothioate-modified RNA, an aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof). The invention also includes methods of using the binding proteins (e.g., antibodies) of the invention to detect and/or quantify the level of a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule) in a sample, and to isolate and/or purify the TAT peptide or TAT fusion molecule present in a mixture. In some embodiments, the TAT fusion molecule comprises a TAT protein transduction domain covalently linked to a cargo moiety. In some embodiments, the cargo moiety is a polypeptide, e.g., an ataxin polypeptide.
In some embodiments, the cargo moiety is a pharmacologically active compound, a small molecule, a liposome encapsulating protein, a radionuclide or radionuclide-labeled compound, a nucleic acid (e.g., siRNA, shRNA, miRNA, phosphorothioate-modified RNA, an aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof). In some embodiments, the methods of the invention further comprise assessing the stability of the TAT fusion molecule.
The invention also includes methods of using the binding proteins (e.g., antibodies) of the invention to inhibit translocation of TAT fusion molecules across cell membranes and to inhibit activity of HIV-TAT protein in a subject by determining the presence of TAT protein or a portion of TAT protein in a biological sample. The invention also includes methods of diagnosing HIV infection in a subject using the binding proteins (e.g., antibodies) of the invention.
I. Definition of
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. However, the meaning and scope of terms should be clear, and the definitions provided herein take precedence over any dictionary or external definition if there is any potential ambiguity. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms, such as "comprises" and "comprising," is not limiting. In addition, unless otherwise specified, terms such as "element" or "component" include both elements and components that comprise one unit and elements and components that comprise more than one subunit. The term "for example" is used herein to mean, and is used interchangeably with, the phrase "for example, but not limited to".
The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.
Unless specifically stated or otherwise clear from the context, the term "about" as used herein is to be understood as being within the normal tolerance of the art, e.g., within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. All numerical values provided herein can be modified by the term "about" unless otherwise expressly stated from the context.
Recitation of a list of chemical groups in the definition of any variable herein includes the definition of that variable as any single group or combination of groups listed. Recitation of embodiments of variables or aspects herein includes embodiments taken as any single embodiment or in combination with any other embodiments or portions thereof.
Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subranges from 1, 2, 3,4, 5,6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 in the group. As used herein, "one or more" is to be understood as each value of 1, 2, 3,4, 5,6, 7, 8, 9,10 and any value greater than 10.
Generally, nomenclature and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed in the present specification. Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The nomenclature used herein and the laboratory procedures and techniques related to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery and treatment of patients.
In order that the present invention may be more readily understood, selected terms are defined as follows.
As used herein, the term "polypeptide" or "peptide" refers to any polymeric chain of amino acids. The term "protein" is used interchangeably with the terms peptide and polypeptide, and also refers to a polymeric chain of amino acids. The term "peptide" or "polypeptide" includes natural or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide may be monomeric or polymeric.
The term "isolated protein" or "isolated polypeptide" or "isolated peptide" is a protein or polypeptide that is not related to its naturally-associated component with which it is associated in its natural state due to its source or derivative source; substantially free of other proteins from the same species; expressed by cells from different species; or not naturally occurring. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived will be "isolated" from its naturally associated components. Proteins may also be substantially free of their naturally associated components by isolation using protein purification techniques well known in the art. An example of an isolated polypeptide is an isolated antibody or antigen-binding portion thereof.
The terms "TAT peptide" or "transactivator of transcription peptides" or "TAT" are used interchangeably herein to refer to transactivating regulatory proteins or portions thereof. The TAT peptide is encoded by the lentivirus HIV-1. TAT peptides are required for efficient transcription of the viral genome (Green and Lowenstein,1988cell 55 (6): 1179-88). The full-length protein comprises 86-101 amino acids, depending on the subtype. In some embodiments, the full length TAT protein has an amino acid sequence as shown in SEQ ID NO 24, as shown below.
Figure BDA0003871364320000111
In vivo, TAT increases the level of transcription of HIV dsDNA. Before TAT is present, a small number of RNA transcripts are produced, allowing production of TAT protein. TAT then binds to cytokines and mediates their phosphorylation, resulting in increased transcription of all HIV genes, providing a positive feedback cycle. TAT also appears to play a more direct role in the course of HIV disease. The TAT protein is released from infected cells in culture and is found in the blood of HIV-1 infected patients.
TAT proteins are capable of translocating through the cytoplasmic membrane and reaching the nucleus to transactivate the viral genome. "TAT protein transduction domains" have been implicated in cell penetration (Vives et al, J Biol chem.1997Jun 20 (25): 16010-7, the contents of which are incorporated herein by reference).
As used herein, the terms "TAT protein transduction domain", "TAT-PTD" or "TAT translocation peptide" are used interchangeably herein and refer to the TAT protein domain of amino acids 47-57 (amino acids YGRKKRRQRRR, SEQ ID NO: 23) of a TAT protein comprising 86 amino acids (Frankel et al, cell, vol.55, issue 6,1988 M.Green et al, cell, vol.55, issue 6,1988 Fawell et al, PNAS,91 (2), 664-668,1994 USPN 6,348,185; the contents of which are incorporated herein by reference. In one embodiment, TAT peptide antibodies of the invention specifically bind to a TAT protein transduction domain. In one embodiment, the TAT protein transduction domain comprises the amino acid sequence of SEQ ID NO:23 (YGRKKRRQRRR). In some embodiments, a TAT peptide binding protein of the invention specifically binds to a TAT protein transduction domain comprising the amino acid sequence of SEQ ID No. 23. In another embodiment, a TAT peptide binding protein of the invention specifically binds to a TAT protein transduction domain consisting essentially of the amino acid sequence of SEQ ID NO. 23. In another embodiment, a TAT peptide binding protein of the invention specifically binds to a TAT protein transduction domain (e.g., SEQ ID NO: 23) comprised in a TAT fusion molecule. For example, in one embodiment, a TAT fusion molecule comprises a TAT-ataxin fusion molecule.
As used herein, "TAT activity" or "TAT peptide activity" includes, but is not limited to, modulating (e.g., increasing) transcription of the viral genome, increasing the level of transcription of HIV dsDNA, modulating phosphorylation of cytokines, and translocation of TAT through the plasma membrane.
As used herein, the term "TAT fusion molecule" or "TAT fusion protein" refers to a TAT peptide, e.g., a TAT protein transduction domain, fused to a cargo moiety. As used herein, the term "cargo moiety" refers to any molecule that can be transported into a cell when fused (e.g., covalently linked) to a TAT peptide (e.g., a TAT protein transduction domain). Thus, the cargo moiety is different from the TAT peptide or any fragment thereof. In one embodiment, the cargo portion is transported in a non-pore forming manner. In one embodiment, the cargo moiety is a polypeptide. In one embodiment, the TAT fusion molecule is a TAT-ataxin fusion molecule and the cargo moiety is an ataxin polypeptide. Exemplary TAT-ataxin fusion molecules are described, for example, in PCT/US2020/044069, which is incorporated herein by reference in its entirety. In another embodiment, the cargo moiety is a therapeutic protein selected from the group consisting of: abarelix, aberra, abciximab, adalimumab, aflibercept, acangase beta, abiirudin, aldesleukin, alfapsepine, alemtuzumab, arabinosidase alpha, aleurizumab, aliskiren, alpha-1-proteinase inhibitor, alteplase, anakinra, acesulfame (acestim), anitipase, anthrax human immunoglobulin, antihemophilic factor, antihemophilic complex, antithrombin alpha, human antithrombin III, antithymotryocytocin, antithymocytocin (horse), antithymocytocin (rabbit), aprotinin, acimumab, asidase alpha (Asfotase Alfa), asparaginase, erwinia Asparaginase (asaginea) Asparaginase alemtuzumab, autologous cultured chondrocytes, basiliximab, becaplamine, berasipu, belimumab, beractant (Beractant), bevacizumab, bivalirudin, bornauzumab, botulinum toxin type a, botulinum toxin type B, brentuximab, brodabitumumab, buserelin, C1 esterase inhibitor (human), cl esterase inhibitor (recombinant), canakinumab, carpumabu, certuzumab, cetuximab, chorionic gonadotropin alpha, chorionic gonadotropin (human), chorionic gonadotropin (recombinant), coagulation factor IX, coagulation factor VIIa, human coagulation factor X, coagulation factor XIII a-subunit (recombinant), collagenase, alfa kenetta (consfa), corticotropin, corticotropin (Cosyntropin), daclizumab, daptomycin, darunavir, bebepoetin alpha, defibrotide (Defibrotide), dineburnine, dinolizumab, dessicin, digoxin immune Fab (ovine), dituximab, alfa streptokinase (Dornase alfa), tegaserod alpha (Drotrecogin alfa), dolabra, eculizumab, efuzumab, eflomuzumab alpha (Efmoroctotog alfa), eloxatase alpha (Elosulfase alfa), eloxazumab, enfuviruzumab, epoetin alpha, epoetin delta, epti, etanercept, elozumab, exenatide, factor IX complex (human), fibrinogen concentrate (human), fibrinolysin (plasmin), filgrastim, etc filgrastim-sndz, follitropin alpha, follitropin beta, sulfatase, intragastric factor, gemtuzumab ozolomicin, glatiramer acetate, recombinant glucagon, glucosidase (Glucarpidase), golimumab, gramicidin D, hepatitis A vaccine, hepatitis B immunoglobulin, human calcitonin, human clostridium tetani toxoid immunoglobulin, human rabies virus immunoglobulin, human Rho (D) immunoglobulin, human serum albumin, human varicella zoster immunoglobulin, hyaluronidase (human recombinant), ibritumomab tikitamurtimae, idazuzumab, idursulfase, imiglucerase, human immunoglobulin, infliximab, insulin aspart, insulin, gemtuzumab ozolomicin, glatiramer acetate, recombinant glucagon, glucosidase, golimumab, gramicidin D, hepatitis A vaccine, hepatitis B immunoglobulin, human calcitonin, human clostridium tetani toxoid immunoglobulin, human rabies virus immunoglobulin, human Rho (D) immunoglobulin, human serum albumin, human varicella zoster immunoglobulin, hyaluronidase (human recombinant), ibumab, ibritumomab, idalizumab, idoxuridine, human immunoglobulin, infliximab, insulin aspart, bovine Insulin, degluin, insulin detemir, insulin glargine, insulin glulisine, insulin lispro, insulin suis, insulin vulgaris, insulin (porcine), insulin isophorne (Insulin-isophane) interferon alpha-2 a recombinant form, interferon alpha-2 b, interferon alfacon-1, interferon alpha-nl, interferon alpha-n 3, interferon beta-la, interferon beta-lb, interferon gamma-lb, intravenous immunoglobulin, prolactin, eculizumab, eprinozumab, larinolase, legumine, lepirrilysin, liraglutin, lucinatant, recombinant human luteinizing hormone (lutropina alfa), mecamylamine, gonadotropin, meprolimus, methoxypolyethylene glycol-epoetin beta, metriptine, moromomonad, natalizumab, natural alpha interferon or multiferron, tolitumomab, nesiritide, nivolumab, obizumab, obituximab, obituzumab ozotan, oxerbitumumab (oxicrastin), plasmin (oipsin), ofatumumab, omalizumab, ompreleukin, ospA lipoprotein, oxytocin, palivimine, palivizumab, pancrelipase, panitumumab, nipagase, pegaptanib, pemetrexed, pefilgrastim, peginterferon alpha-2 a, peginterferon alpha-2 b, peginterferon beta-1 a, pegolozyme, pegvisomant, pembropamumab, pertuzumab, swine lung phospholipid (Poractant Alfa), pramine peptide, pretact, protamine sulfate, human protein S, prothrombin complex concentrate, ragweed pollen extract, ramucirumumab, ranibizumab, labrasilase, ranibixib, reteplase, rilonaxipu, rituximab, romidepsin, glycosidase, salmon calcitonin, sarsashimubumab, sartuzumab (samotuzumab) Sebelipase Alfa, secretin, secukinumab, semorelin (Sermorelin), serum albumin, iodinated serum albumin, setuximab, semoclotucin alpha (Simocog Alfa), sipuleucel-T, growth hormone recombinant, streptokinase, sulodexide, recombinant protein susogelysin alpha (Susoctocog Alfa), talosidase alpha, teduglutide, teicoplanin, tenenaproxen, teriparatide, tesamolin, thrombomodulin, thymosin, thyroglobulin, thyrotropin alpha, tollizumab, tositumomab, trastuzumab, tuberculin purified protein derivatives, tolterodine alpha (Turocog Alfa), urofollitropin, urokinase, ulteckelekin, vasopressin, vildazumab, vilamina, thrombomodulin alpha, thymine, thyroglobulin, thyroid hormone, tosituzumab, tositumumab, trastuzumab, tuberculin purified protein derivatives, tolocrelidin alpha (Turococog alfa), urofollitropin, urokinase, ultecumab, vasopressin, victorizumab, and vildaglase alpha. In another embodiment, the protein is selected from the group consisting of those included in Raghava, gajendra p.s.; usmani, salman Sadullah; bedi, gursimura; samuel, jesse S.; singh, sandep; kalra, sourav; et al (2017) THPdb: those of Database of FDA applied Peptide and Protein Therapeutics, the contents of which are incorporated herein by reference.
In one embodiment, the size of the polypeptide cargo moiety may be between about 100kD and 200 nM. In another embodiment, the cargo moiety is a pharmacologically active compound, a small molecule, a liposome encapsulating protein, a radionuclide or radionuclide-labeled compound, a nucleic acid (e.g., siRNA, shRNA, miRNA, phosphorothioate-modified RNA, an aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof). In another embodiment, the TAT fusion molecule is a tissue penetrating TAT fusion molecule.
The term "TAT-ataxin fusion molecule" as used herein includes any fusion molecule comprising a TAT protein transduction domain or fragment thereof and an ataxin peptide or fragment thereof. In one embodiment, fusion of the ataxin polypeptide to a TAT peptide (e.g., a TAT protein transduction domain) allows translocation of the entire fusion protein across the cell membrane. Exemplary TAT-ataxin fusion molecules are described in U.S. Pat. No. 8,283,444, the contents of which are incorporated herein by reference.
As used herein, "detecting," "determining," and the like are to be understood as assays that are performed to recognize a particular antigen (e.g., a TAT protein transduction domain or a TAT fusion molecule) (e.g., a TAT-ataxin fusion molecule) in a sample. Detection may be by any method known in the art. For example, a TAT protein transduction domain or TAT fusion molecule (e.g., a TAT-ataxin fusion molecule) can be detected by contacting a sample comprising a TAT protein transduction domain or TAT fusion molecule with a binding protein under conditions such that the binding protein of the invention binds to the TAT protein transduction domain in the sample. In one embodiment, the binding protein is immobilized. In another embodiment, the TAT transduction domain or TAT fusion molecule is eluted from the immobilized binding protein.
As used herein, the term "specifically binds" or "specifically binds" with respect to the interaction of a binding protein (e.g., an antibody, protein or peptide) with a second chemical means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical; for example, antibodies recognize and bind to specific protein structures rather than to common proteins. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.
In one embodiment, as used herein, the phrase "specifically binds to" or "specifically binds to" a TAT protein transduction domain refers to the ability of an anti-TAT binding protein to interact with the TAT protein transduction domain, the dissociation constant (K) D ) About 2,000nM or less, about 1,000nM or less, about 500nM or less, about 200nM or less, about 100nM or less, about 75nM or less, about 50nM or less, about 43nM or less, about 25nM or less, about 20nM or less, about 19nM or lessLow, about 18nM or less, about 17nM or less, about 16nM or less, about 15nM or less, about 14nM or less, about 13nM or less, about 12nM or less, about 11nM or less, about 10nM or less, about 9nM or less, about 8nM or less, about 7nM or less, about 6nM or less, about 5nM or less, about 4nM or less, about 3nM or less, about 2nM or less, about 1nM or less, about 0.5nM or less, about 0.3nM or less, about 0.1nM or less, or about 0.01nM or less, or about 0.001nM or less.
In another embodiment, as used herein, the phrase "specifically binds to" or "specifically binds to" a TAT protein transduction domain refers to the ability of an anti-TAT binding protein to interact with the TAT protein transduction domain, the dissociation constant (K) D ) Between about 1pM (0.001 nM) and 2,000nM, between about 500pM (0.5 nM) and 1,000nM, between about 500pM (0.5 nM) and 500nM, between about 1nM and 200nM, between about 1nM and 100nM, between about 1nM and 50nM, between about 1nM and 20nM, or between about 1nM and 5 nM. In one embodiment, K is determined by surface plasmon resonance D 。
As used herein, the term "antibody" broadly refers to any immunoglobulin (Ig) molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof, which retains the essential epitope binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody forms are known in the art. Non-limiting embodiments of which are discussed below.
In a full-length antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain CL. The VH and VL regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecules can be of any type (e.g., igG, igE, igM, igD, igA, and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass. In a preferred embodiment, the immunoglobulin molecule is an IgG1.
As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., a TAT protein transduction domain). It has been shown that the antigen binding function of an antibody can be achieved by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, bispecific or multispecific; specifically binds to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et Al, (1989) Nature341:544-546, winter et Al, PCT publication No. WO 90/05144Al, incorporated herein by reference), which comprise a single variable domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., bird et al, (1988) Science 242 423-426; and Huston et al, (1988) Proc. Natl. Acad. Sci. USA 85 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. In certain embodiments, the scFv molecule can be incorporated into a fusion protein. Other forms of single chain antibodies (e.g., diabodies) are also included. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and creating two antigen binding sites (see, e.g., holliger, p. Et al, (1993) proc.natl.acad.sci.usa 90 6444-6448 poljak, r.j. Et al, (1994) Structure2: 1121-1123. Such Antibody-binding moieties are known in the art (Kontermann and Dubel eds., antibody Engineering (2001) Springer-Verlag. New York.790pp. (ISBN 3-540-41354-5)).
As used herein, the term "antibody construct" refers to a polypeptide comprising one or more antigen-binding portions disclosed herein linked to a linker polypeptide or an immunoglobulin constant region. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding moieties. Such linker polypeptides are well known in the art (see, e.g., holliger, p. Et al, (1993) proc.natl.acad.sci.usa 90 6444-6448, poljak, r.j., et al, (1994) Structure 2. Immunoglobulin constant regions refer to heavy or light chain constant regions. Antibody portions (e.g., fab and F (ab') 2 Fragments) can be prepared from whole antibodies using conventional techniques, e.g., papain or pepsin digestion of whole antibodies, respectively. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
As used herein, an "isolated binding protein" or "isolated antibody" refers to a binding protein that is substantially free of other binding proteins having different antigenic specificities, e.g., an antibody (e.g., an isolated antibody that specifically binds to a TAT protein transduction domain is substantially free of antibodies that specifically bind antigens other than a TAT protein transduction domain). Furthermore, the isolated binding protein may be substantially free of other cellular material and/or chemicals.
The term "humanized antibody" refers to antibodies that comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse), but in which at least a portion of the VH and/or VL sequences have been altered to be more "human-like," i.e., more similar to human germline variable region sequences. In particular, the term "humanized antibody" is an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest, comprising an amino acid sequence substantially having a human antibodyAnd a Complementarity Determining Region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of CDRs refers to CDRs having an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. Humanized antibodies comprise substantially all, at least one, and usually two, variable domains (Fab, fab ', F (ab') 2 FabC, fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody), and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody comprises a light chain and at least the variable domain of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In other embodiments, the humanized antibody comprises only humanized heavy chains. In particular embodiments, the humanized antibody comprises only a humanized variable domain of a light chain and/or a humanized heavy chain.
The humanized antibody may be selected from any class of immunoglobulin including IgM, igG, igD, igA and IgE and any isotype including but not limited to IgG1, igG2, igG3 and IgG4. Humanized antibodies may comprise sequences from more than one class or isotype, and specific constant regions may be selected to optimize desired effector function using techniques well known in the art.
The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms are recognized in the art as referring to the numbering system of amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof (Kabat et al, (1971) Ann. NY Acad, sci.190:382-391 and Kabat, E.A., et al, (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 of CDR1, amino acid positions 50 to 65 of CDR2 and amino acid positions 95 to 102 of CDR 3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 of CDR1, amino acid positions 50 to 56 of CDR2 and amino acid positions 89 to 97 of CDR 3.
As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are three CDRs in each variable region of the Heavy (HC) and Light (LC) chains, which are named CDR1, CDR2 and CDR3 (or specifically HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR 3) for each variable region. As used herein, the term "set of CDRs" refers to a set of three CDRs that occur from a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined differently from system to system. The system described by Kabat (Kabat et al, sequences of Proteins of Immunological Interest (CDR), md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries defining the three CDRs, which CDRs may be referred to as Kabat CDRs, chothia and coworkers (Chothia & Lesk, J.mol.biol.196:901-917 (1987) and Chothia et al, nature 342.
As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region from which the CDRs are removed. Since the exact definition of the CDR sequences can be determined by different systems, the meaning of the framework sequences also has correspondingly different interpretations. The 6 CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain and CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into 4 subregions (FR 1, FR2, FR3 and FR 4) on each chain, with CDR1 being located between FR1 and FR2, CDR2 being located between FR2 and FR3 and CDR3 being located between FR3 and FR4. Without defining a particular sub-region as FR1, FR2, FR3 or FR4, as otherwise mentioned, the framework region represents a combination of FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR denotes one of the four sub-regions, and FRs denotes two or more of the four sub-regions constituting the framework region.
The framework regions and CDR regions of the humanized antibody need not correspond exactly to the parental sequences, e.g., the donor antibody CDR or consensus framework can be mutagenized by substitution, insertion, and/or deletion of at least one amino acid residue such that the CDR or framework residue at that position does not correspond to the donor antibody or consensus framework. However, in preferred embodiments, such mutations are not extensive. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework regions in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed From the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., winnaker, from Genes to Clones (Verlagsgesellschaft, weinheim, germany 1987.) in an immunoglobulin family, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the family.
"percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without regard to any conservative substitutions of moieties that are considered to be identical in the sequence. Alignment for the purpose of determining percent amino acid identity can be accomplished in a variety of ways known to those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.
The term "multivalent antibody" is used herein to refer to an antibody comprising two or more antigen binding sites. In certain embodiments, multivalent antibodies can be engineered to have three or more antigen binding sites, and are not typically naturally occurring antibodies.
The term "multispecific antibody" refers to an antibody that is capable of binding two or more unrelated antigens.
The terms "dual variable domain" or "DVD" are used interchangeably herein, are antigen binding proteins that comprise two or more antigen binding sites, and are tetravalent or multivalent binding proteins. Such DVDs can be monospecific (i.e., capable of binding one antigen) or multispecific (i.e., capable of binding two or more antigens). A DVD-binding protein comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides is referred to as DVD Ig. Each half of the DVD Ig contains a heavy chain DVD polypeptide, a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable region and a light chain variable region with a total of 6 CDRs involved in antigen binding of each antigen binding site. In one embodiment, the CDRs described herein are used for anti-TAT DVD.
The term "activity" includes the activity of a binding protein (e.g., an antibody) with respect to an antigen, e.g., binding specificity/affinity, e.g., the activity of an anti-TAT-antibody binding to a TAT protein transduction domain antigen. In one embodiment, anti-TAT antibody activity includes, but is not limited to, binding to a TAT protein transduction domain in vitro; binding to a TAT protein transduction domain in vivo; and reducing or inhibiting HIV infection.
The term "epitope" refers to a region of an antigen that is bound by a binding protein (e.g., an antibody or antibody portion). In certain embodiments, epitopic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have particular three-dimensional structural characteristics and/or particular charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration within a Biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, uppsala, sweden and Piscataway, NJ). For further description, see
Figure BDA0003871364320000231
U.S. et al, (1993) Ann.biol.Clin.51:19-26;
Figure BDA0003871364320000232
U.S. et al, (1991) Biotechniques 11; johnsson, B, et al, (1995) J.mol.Recognit.8:125-131; and Johnnson, B.et al, (1991) anal. Biochem.198:268-277.
As used herein, the term "k on "or" k a "refers to the binding rate constant of an antibody to an antigen to form an antibody/antigen complex.
As used herein, the term "k off "or" k d "refers to the dissociation rate constant of an antibody from an antibody/antigen complex.
As used herein, the term "K D "refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. K D From k to k a /k d And (4) calculating.
As used herein, the term "competitive binding" refers to the situation where a first antibody competes with a second antibody for a binding site on a third molecule (e.g., an antigen). In one embodiment, the competitive binding between the two antibodies is determined using FACS analysis.
The term "competitive binding assay" is an assay for determining whether two or more antibodies bind to the same epitope. In one embodiment, the competitive binding assay is a competitive entangle light activated cell sorting (FACS) assay used to determine whether two or more antibodies bind to the same epitope by determining whether the entangle light signal of a labeled antibody is reduced by the introduction of a non-labeled antibody, wherein competition for the same epitope would reduce the entangle light level.
The term "labeled binding protein" as used herein refers to a binding protein, such as an antibody, that is incorporated with a label that provides for recognition of the binding protein or target moiety. Preferably, the label is a detectable label, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotin moiety detectable by labeled avidin (e.g., streptavidin comprising a fluorescent label or enzymatic activity detectable by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: a radioisotope or radionuclide (e.g., 3 H、 14 C、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I、 177 Lu、 166 ho or 153 Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, fluorescent cellulase, alkaline phosphatase); a chemiluminescent label; a biotin group; biotin, digoxigenin, a predetermined polypeptide epitope recognized by a second reporter (e.g., leucine zipper pair sequence, binding site of a second antibody, metal binding domain, epitope tag); and magnetic agents such as gadolinium chelates.
The term "polynucleotide" as used herein refers to a polymeric form of two or more nucleotides (ribonucleotides or deoxyribonucleotides, or a modified form of either nucleotide). The term includes single-stranded and double-stranded forms of DNA, but preferably double-stranded DNA.
As used herein, the term "isolated polynucleotide" shall mean a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof) that, due to its origin,: is not related to all or part of a polynucleotide of an "isolated polynucleotide" found in nature; operably linked to a polynucleotide that is not linked in nature; or not as part of a larger sequence in nature.
The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Exemplary vectors include, for example, pcDNA, pTT3, pEFBOS, pBV, pJV, pBJ, pGEX, VSV, pBR322, pCMV-HA, pEN, YAC, BAC, phage lambda, phagemid, pCAS9, pCEN6, pYESIL, p3HPRTl, pFN2A, pBC, pTZ, pGEM, pGEMK, pEX, pSAR, pCEP, cosmid, pBluescript, pKK, pFLoxin, pCP, pHR, pUC, and pMAL. Other plasmids are included in international publication No. WO2005108568, the contents of which are incorporated herein by reference.
The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "operably linked" sequences include expression control sequences that are contiguous with the gene of interest and that act in trans or at a distance to control the gene of interest. As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary to affect the expression and processing of the coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., kozak consensus sequence); sequences that enhance protein stability; and, when desired, sequences that enhance protein secretion. The nature of such control sequences varies from host organism to host organism; in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and may also include other components whose presence is advantageous, such as leader sequences and fusion partner sequences. The protein constructs of the invention can be expressed and purified using expression vectors and host cells known in the art, including expression cassettes, vectors, recombinant host cells, and methods for recombinant expression and proteolytic processing of recombinant polyproteins and proproteins from a single open reading frame (e.g., WO2007/014162, which is incorporated herein by reference).
As defined herein, "transformation" refers to any procedure by which foreign DNA enters a host cell. Transformation can be performed under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method of inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. The method is selected according to the host cell to be transformed, and may include, but is not limited to, viral infection, electroporation, lipofection, and biolistic methods. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication as an autonomously replicating plasmid or as part of the host chromosome. They also include cells that transiently express the inserted DNA or RNA for a limited period of time.
As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which exogenous DNA has been introduced. It is understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Preferably, the host cell comprises a prokaryotic and eukaryotic cell selected from any of the kingdoms of life. Preferred eukaryotic cells include protists, fungi, plant and animal cells. Most preferably, host cells include, but are not limited to, the prokaryotic cell line E.coli; mammalian cell lines CHO, HEK 293 and COS; insect cell line Sf9; and fungal cells, saccharomyces cerevisiae.
Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods well known in the art and described in various comprehensive and more specific references cited and discussed in the present specification. See, e.g., sambrook et al, molecular Cloning: A Laboratory Manual (2 d ed., cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
As used herein, the term "sample" is used in its broadest sense. As used herein, "biological sample" includes, but is not limited to, any amount of material from a living organism or a previously living organism. These living organisms include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits, and other animals. These substances include, but are not limited to, blood, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.
A "patient" or "subject" to be treated by the methods of the invention may refer to a human or non-human animal, preferably a mammal. By "subject" is meant any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds. The human subject may be referred to as a patient. It should be noted that the clinical observations described herein are made on human subjects, and in at least some embodiments, the subjects are humans.
The terms "disorder" and "disease" are used inclusively and refer to any deviation in any part, organ or system of the body (or any combination thereof) from normal structure or function. A particular disease manifests itself as characteristic symptoms and signs, including biological, chemical, and physical changes, and is often associated with a variety of other factors, including but not limited to demographic, environmental, employment, genetic, and medical history factors. Certain characteristic signs, symptoms and related factors can be quantified by a variety of methods to yield important diagnostic information. As used herein, "TAT-related disease" includes Human Immunodeficiency Virus (HIV) infection or acquired immunodeficiency syndrome (AIDS) and symptoms caused by or associated with HIV infection or AIDS.
As used herein, the term "human immunodeficiency virus" or "HIV" refers generally to retroviruses, which are causative agents of acquired immunodeficiency syndrome (AIDS), variants thereof (e.g., simian acquired immunodeficiency syndrome, SAIDS), and diseases, conditions, or opportunistic infections associated with AIDS or variants thereof, and includes HIV-type 1 (HIV-1) and HIV-type 2 (HIV-2), related retroviruses (e.g., simian Immunodeficiency Virus (SIV)) and variants thereof (e.g., engineered retroviruses, such as chimeric HIV viruses, e.g., simian immunodeficiency virus (SHIV)) of any clade or strain therein. The former names of HIV include human T-lymphotropic virus Ill (HTLV-III), lymphadenopathy-associated virus (LAV) and AIDS-Associated Retrovirus (ARV).
As used herein, and as is well known in the art, "treatment" is a method for obtaining beneficial or desired results (e.g., clinical results). Beneficial or desired results can include, but are not limited to, cure or eradication of a disease, disorder, or condition (e.g., HIV infection); reduction or amelioration of one or more symptoms or conditions (e.g., HIV infection); reduction in the extent of a disease, disorder, or condition (e.g., HIV infection); stabilization (i.e., not worsening) of the state of the disease, disorder, or condition (e.g., HIV infection); prevention of the spread or dissemination of a disease, disorder, or condition (e.g., HIV infection); delay or slowing of progression of a disease, disorder, or condition (e.g., HIV infection); amelioration or palliation of the disease, disorder or condition (e.g., HIV infection); and mitigation (partial or total), whether detectable or not.
As used herein, "curing" a subject (e.g., a human) infected with a retrovirus (e.g., human Immunodeficiency Virus (HIV) (e.g., HIV type 1 or HIV type 2)) refers to obtaining and maintaining virologic control in the absence of antiretroviral therapy (ART) for at least two months (e.g., 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, or5 years or more).
The term "expression" as used herein refers to the process of producing a polypeptide from DNA. The process involves transcription of a gene into mRNA and translation of the mRNA into a polypeptide. Depending on the context of use, "expression" may refer to the production of RNA or protein or both.
As used herein, the term "obtained" is understood herein to mean manufactured, purchased, or otherwise possessed.
Reference will now be made in detail to exemplary embodiments of the invention. While the invention will be described in conjunction with the exemplary embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention as defined by the appended claims.
Binding proteins binding to TAT peptides
One aspect of the invention provides an isolated binding protein, e.g., an antibody or antigen-binding portion thereof, that binds to a TAT peptide and specifically binds to a TAT protein transduction domain. anti-TAT peptide antibodies are also described in detail below, as well as methods of making binding proteins, methods of producing binding proteins.
A. anti-TAT peptide binding proteins
The invention features binding proteins, e.g., antibodies, that include an antigen binding domain, the binding proteins capable of binding to a TAT peptide. In particular, the binding protein is capable of binding to a TAT protein transduction domain. Novel antibodies are collectively referred to herein as "TAT peptide antibodies" or "anti-TAT antibodies".
Although the term "antibody" is used throughout, it should be noted that antibody portions (i.e., antigen-binding portions of anti-TAT antibodies) are also included within the present disclosure and may be included in the embodiments (methods and compositions) described throughout. For example, an anti-TAT antibody moiety may be conjugated to a drug, as described herein. In certain embodiments, the anti-TAT antibody binding portion is a Fab, fab ', F (ab') 2 Fv, disulfide-linked Fv, scFv, single domain antibody or diabody.
A list of the amino acid sequences of the VH and VL regions and CDRs of a preferred monoclonal antibody of the invention is shown in Table 1 of example 1.
In one aspect, the invention features a binding protein comprising an antigen binding domain capable of binding to a TAT protein transduction domain, said antigen binding domain comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 14 and 15; and a light chain variable region comprising an amino acid sequence selected from the group consisting of 5,9, 13, and 19.
In one aspect, the invention features a binding protein comprising an antigen binding domain capable of binding to a TAT protein transduction domain, said antigen binding domain comprising a heavy chain CDR set (CDR 1, CDR2 and CDR 3) selected from those listed in table 1; and a set of LC CDRs (CDR 1, CDR2 and CDR 3) selected from those listed in table 1.
In one aspect, the invention features a binding protein comprising an antigen binding domain capable of binding to a TAT protein transduction domain, the antigen binding domain comprising a heavy chain CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID No. 4 or SEQ ID No. 18. In one embodiment, the binding protein further comprises a heavy chain CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 3 or SEQ ID NO 17. In another embodiment, the binding protein further comprises a heavy chain CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 2 or SEQ ID NO 16. In another embodiment, the binding protein further comprises a light chain CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 8, SEQ ID NO 12, or SEQ ID NO 22. In another embodiment, the binding protein further comprises a light chain CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 7, SEQ ID NO 11 or SEQ ID NO 21. In another embodiment, the binding protein further comprises a light chain CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 6, SEQ ID NO 10 or SEQ ID NO 20.
In another aspect, the invention features a binding protein comprising an antigen binding domain capable of binding to a TAT protein transduction domain, the antigen binding domain comprising: a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence shown in SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence shown in SEQ ID NO. 3 and a CDR1 domain comprising the amino acid sequence shown in SEQ ID NO. 2, or a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence shown in SEQ ID NO. 18, a CDR2 domain comprising the amino acid sequence shown in SEQ ID NO. 17 and a CDR1 domain comprising the amino acid sequence shown in SEQ ID NO. 16; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence shown in SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence shown in SEQ ID NO. 7 and a CDR1 domain comprising the amino acid sequence shown in SEQ ID NO. 6, or a light chain variable region comprising a CDR3 domain comprising the amino acid sequence shown in SEQ ID NO. 12, a CDR2 domain comprising the amino acid sequence shown in SEQ ID NO. 11 and a CDR1 domain comprising the amino acid sequence shown in SEQ ID NO. 10, or a light chain variable region comprising a CDR3 domain comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 domain comprising the amino acid sequence shown in SEQ ID NO. 21 and a CDR1 domain comprising the amino acid sequence shown in SEQ ID NO. 20.
In other aspects, the invention also features a binding protein comprising an antigen binding domain capable of binding to a TAT protein transduction domain, the antigen binding domain comprising: a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3 and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7 and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6.
In other aspects, the invention also features a binding protein comprising an antigen binding domain capable of binding to a TAT protein transduction domain, the antigen binding domain comprising: a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3 and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 11 and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 10.
In other aspects, the invention also features a binding protein comprising an antigen binding domain capable of binding to a TAT protein transduction domain, the antigen binding domain comprising: a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 18, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 17 and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 16; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO:22, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:21 and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 20.
In a further embodiment, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 5.
In another further embodiment, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 9.
In another further embodiment, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 13.
In another further embodiment, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 14 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 5.
In another further embodiment, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 15 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 19.
In certain embodiments, the term "10-1" refers to a hybridoma that produces an antibody comprising (i) a variable heavy chain having an amino acid sequence comprising SEQ ID NO: 1; and (ii) a variable light chain having an amino acid sequence comprising SEQ ID NO: 5. In certain embodiments, the 10-1 heavy chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2, and the light chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6. In certain embodiments, antibody 10-1 can have at least about 1 x 10 as measured by surface plasmon resonance 4 M -1 s -1 To about 6X 10 6 M -1 s -1 Binding to TAT protein transduction DomainRate constant (K) ON ). In other embodiments, a binding protein according to the invention can have a size of at least about 2.7 x 10 as measured by surface plasmon resonance 5 M -1 s -1 Of (a) an association rate constant (K) to a TAT protein transduction domain ON ). In other embodiments, a binding protein according to the invention may have a size of 1.0 × 10 -12 s -1 Or a dissociation constant (K) for TAT protein transduction domain of lower D ). In certain preferred embodiments, the binding proteins of the invention have a size of about 1.0X 10 -7 s -1 Or lower; 1.0X 10 -8 s -1 Or lower; 1.0X 10 -9 s -1 Or lower; 1.0X 10 -10 s -1 Or lower; 1.0X 10 -11 s -1 Or lower; and 1.0X 10 -12 s -1 Or a lower dissociation constant (K) for a TAT protein transduction domain D ). According to a preferred embodiment of the invention, the isotype of the antibody construct produced by the 10-1 hybridoma clone is IgG1K.
In some embodiments, the anti-TAT antibody, or antigen-binding portion thereof, comprises: a heavy chain comprising the amino acid sequence shown as SEQ ID NO. 1 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1, and/or a light chain comprising the amino acid sequence shown as SEQ ID NO. 5 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 5.
In certain embodiments, the term "10-12" refers to a hybridoma that produces an antibody comprising (i) a variable heavy chain having an amino acid sequence comprising SEQ ID NO: 14; and (ii) a variable light chain having an amino acid sequence comprising SEQ ID NO: 5. In certain embodiments, the 10-12 heavy chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2, and the light chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6. In some embodimentsIn one embodiment, antibodies 10-12 can have a size of at least about 1X 10 as measured by surface plasmon resonance 4 M -1 s -1 To about 6X 10 6 M -1 s -1 Of (a) an association rate constant (K) to a TAT protein transduction domain ON ). In other embodiments, a binding protein according to the invention can have a size of at least about 2.7 x 10 as measured by surface plasmon resonance 5 M - 1 s -1 The association rate constant (K) to the TAT protein transduction domain of (1) ON ). In other embodiments, a binding protein according to the invention may have a size of 1.0 × 10 -12 s -1 Or a dissociation constant (K) for TAT protein transduction domain of lower D ). In certain preferred embodiments, a binding protein according to the invention has a size of about 1.0X 10 -7 s -1 Or lower; 1.0X 10 -8 s -1 Or lower; 1.0X 10 -9 s -1 Or lower; 1.0X 10 -10 s -1 Or lower; 1.0X 10 -11 s -1 Or lower; and 1.0X 10 -12 s -1 Or a lower dissociation constant (K) for a TAT protein transduction domain D ). According to a preferred embodiment of the invention, the isotype of the antibody construct produced by the 10-12 hybridoma clone is IgG1K.
In some embodiments, the anti-TAT antibody, or antigen-binding portion thereof, comprises: a heavy chain comprising the amino acid sequence shown as SEQ ID NO. 14 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 14, and/or a light chain comprising the amino acid sequence shown as SEQ ID NO. 5 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 5.
In certain embodiments, the terms "10-4", "10-5", "12-1" and "12-3" refer to hybridomas that produce antibodies comprising (i) a variable heavy chain having an amino acid sequence comprising SEQ ID NO: 1; and (ii) a variable light chain having an amino acid sequence comprising SEQ ID NO 9. In certain embodiments, the 10-4, 10-5, 12-1, and 12-3 heavy chain variable regions comprise a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 4, a CDR containing SEQ ID NO. 43 and a CDR1 domain comprising the amino acid sequence of SEQ ID No. 2, and the light chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID No. 12, a CDR2 domain comprising the amino acid sequence of SEQ ID No. 11 and a CDR1 domain comprising the amino acid sequence of SEQ ID No. 10. In certain embodiments, antibodies 10-4, 10-5, 12-1 and 12-3 can have at least about 1X 10 as measured by surface plasmon resonance 4 M -1 s -1 To about 6X 10 6 M -1 s -1 Of (a) an association rate constant (K) to a TAT protein transduction domain ON ). In other embodiments, a binding protein according to the invention can have a size of at least about 2.7 x 10 as measured by surface plasmon resonance 5 M -1 s -1 Of (a) an association rate constant (K) to a TAT protein transduction domain ON ). In other embodiments, a binding protein according to the invention may have a size of 1.0 × 10 -12 s -1 Or a lower dissociation constant (K) for a TAT protein transduction domain D ). In certain preferred embodiments, a binding protein according to the invention has a size of about 1.0X 10 -7 s -1 Or lower; 1.0X 10 -8 s -1 Or lower; 1.0X 10 -9 s -1 Or lower; 1.0X 10 -10 s -1 Or lower; 1.0X 10 -11 s -1 Or lower; and 1.0X 10 -12 s -1 Or a dissociation constant (K) for TAT protein transduction domain of lower D ). According to a preferred embodiment of the invention, the isotype of the antibody constructs produced by the 10-4, 10-5, 12-1 and 12-3 hybridoma clones is IgG1/K.
In some embodiments, the anti-TAT antibody, or antigen binding portion thereof, comprises: a heavy chain comprising the amino acid sequence shown as SEQ ID NO. 1 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1, and/or a light chain comprising the amino acid sequence shown as SEQ ID NO.9 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 9.
In certain embodiments, the terms "10-9", "12-8" and "12-10" refer to productionA hybridoma producing an antibody comprising (i) a variable heavy chain having an amino acid sequence comprising SEQ ID NO: 1; and (ii) a variable light chain having an amino acid sequence comprising SEQ ID NO 13. In certain embodiments, the 10-9, 12-8, and 12-10 heavy chain variable regions comprise a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2, and the light chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6. In certain embodiments, antibodies 10-9, 12-8, and 12-10 can have at least about 1X 10 as measured by surface plasmon resonance 4 M -1 s -1 To about 6X 10 6 M -1 s -1 The association rate constant (K) to the TAT protein transduction domain of (1) ON ). In other embodiments, a binding protein according to the invention can have a size of at least about 2.7 x 10 as measured by surface plasmon resonance 5 M -1 s -1 Of (a) an association rate constant (K) to a TAT protein transduction domain ON ). In other embodiments, a binding protein according to the invention may have a size of 1.0 × 10 -12 s -1 Or a lower dissociation constant (K) for a TAT protein transduction domain D ). In certain preferred embodiments, a binding protein according to the invention has a size of about 1.0X 10 -7 s -1 Or lower; 1.0X 10 -8 s -1 Or lower; 1.0X 10 -9 s -1 Or lower; 1.0X 10 -10 s -1 Or lower; 1.0X 10 -11 s -1 Or lower; and 1.0X 10 -12 s -1 Or a lower dissociation constant (K) for a TAT protein transduction domain D ). According to a preferred embodiment of the invention, the isotype of the antibody construct produced by the 10-9, 12-8 and 12-10 hybridoma clones is IgG 1 /K。
In some embodiments, the anti-TAT antibody, or antigen-binding portion thereof, comprises: a heavy chain comprising the amino acid sequence shown as SEQ ID NO. 1 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1, and/or a light chain comprising the amino acid sequence shown as SEQ ID NO. 13 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 13.
In certain embodiments, the term "6.3" refers to a hybridoma that produces an antibody comprising (i) a variable heavy chain having an amino acid sequence comprising SEQ ID NO: 14; and (ii) a variable light chain having an amino acid sequence comprising SEQ ID NO: 5. In certain embodiments, the 6.3 heavy chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2, and the light chain variable region comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6. In certain embodiments, antibody 6.3 can have at least about 1 x 10 as measured by surface plasmon resonance 4 M - 1 s -1 To about 6X 10 6 M -1 s -1 The association rate constant (K) to the TAT protein transduction domain of (1) ON ). In other embodiments, a binding protein according to the invention can have a size of at least about 2.7 x 10 as measured by surface plasmon resonance 5 M -1 s -1 Of (a) an association rate constant (K) to a TAT protein transduction domain ON ). In other embodiments, a binding protein according to the invention may have a size of 1.0 × 10 -12 s -1 Or a lower dissociation constant (K) for a TAT protein transduction domain D ). In certain preferred embodiments, a binding protein according to the invention has a size of about 1.0X 10 -7 s -1 Or lower; 1.0X 10 -8 s -1 Or lower; 1.0X 10 -9 s -1 Or lower; 1.0X 10 -10 s -1 Or lower; 1.0X 10 -11 s -1 Or lower; and 1.0X 10 -12 s -1 Or a lower dissociation constant (K) for a TAT protein transduction domain D ). According to a preferred embodiment of the invention, the antibody constructs produced by the 6.3 hybridoma clonesThe isotype of the body is IgG21K.
In some embodiments, the anti-TAT antibody, or antigen-binding portion thereof, comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO. 15 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 15, and/or a light chain comprising the amino acid sequence shown in SEQ ID NO. 19 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 19.
According to a preferred embodiment of the invention, the binding protein described herein is an antibody.
Accordingly, the invention features an antibody construct comprising a binding protein described herein, wherein the antibody construct further comprises a linker polypeptide or an immunoglobulin constant region.
The antibody construct according to the invention may comprise a heavy chain immunoglobulin constant region selected from the group consisting of: an IgM constant region, an IgG4 constant region, an IgG1 constant region, an IgE constant region, an IgG2 constant region, an IgG3 constant region, and an IgA constant region.
In addition, the antibody may comprise a light chain constant region (either a kappa light chain constant region or a lambda light chain constant region).
In certain embodiments, a binding protein according to the invention may have a size selected from the group consisting of about 1 × 10 as measured by surface plasmon resonance 4 M -1 s -1 To about 6X 10 6 M -1 s -1 Group of association rate constants (K) to TAT protein transduction domains ON ). In other embodiments, a binding protein according to the invention can have at least about 2.5 x 10 as measured by surface plasmon resonance 5 M -1 s -1 And at least about 2.7X 10 5 M -1 s -1 Of (a) to the TAT peptide ON )。
In other embodiments, a binding protein according to the invention may have a dissociation constant (K) for a TAT protein transduction domain D ) Selected from the group consisting of: about 1.0X 10 -7 s -1 Or lower; 1.0X 10 -8 s -1 Or lower; 1.0X 10 - 9 s -1 Or lower; 1.0X 10 -10 s -1 Or lower; 1.0X 10 -11 s -1 Or lower; and 1.0X 10 -12 s -1 Or lower. In certain preferred embodiments, the binding protein according to the invention has a size of 1.0 × 10 -7 s -1 Or a lower dissociation constant (K) for TAT peptides D )。
The binding protein may be selected from the group consisting of an immunoglobulin molecule, a monoclonal antibody, a murine antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a single domain antibody, an Fv, a disulfide-linked Fv, an scFv, a diabody, an Fab ', an F (ab') 2 Multispecific antibodies, dual specific antibodies and bispecific antibodies.
Alternatively, the antibody portion may be, for example, a Fab fragment or a single chain Fv fragment.
Substitutions of amino acid residues in the Fc portion to alter antibody effector functions are known in the art (Winter et al, U.S. Pat. No. 5,648,260; no. 5624821). The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement Dependent Cytotoxicity (CDC) and half-life/clearance of the antibody and antigen-antibody complex. In some cases, these effector functions are required for therapeutic antibodies, but in other cases may be unnecessary or even detrimental, depending on the therapeutic objective. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC by binding to Fc γ R and complement C1q, respectively. Neonatal Fc receptor (FcRn) is a key component determining the circulating half-life of antibodies. In another embodiment, at least one amino acid residue in a constant region of an antibody (e.g., the Fc region of an antibody) is substituted such that the effector function of the antibody is altered.
One embodiment provides a labeled binding protein, wherein an antibody or antibody portion of the invention is derivatized with or linked to one or more functional or cargo molecules. In one embodiment, the cargo moiety is another peptide or protein, e.g., an ataxin polypeptide. In one embodiment, the cargo moiety is a polypeptide selected from the group consisting of: abarelix, abamectin, abciximab, adalimumab, aflibercept, acangase beta, abiirudin, aldesleukin, alfapsept, alemtuzumab, arabinosidase alpha, aleurizumab, aliskiren alpha-1-protease inhibitor, alteplase, anakinra, ansamitriptan, anthrax human immunoglobulin, antihemophilin factor, antithrombin complex, antithrombin alpha, human antithrombin III, antithymotrypsin (horse), antithymotrypsin (rabbit), aprotinin, acipimox, asimomab, asprofecox alpha, asparaginase, erwinia Asparaginase (asparaginia chrysophanase), atezumab arguase, autologous cultured chondrocytes basiliximab, becaplamine, berasipu, belimumab, beliracetam, bevacizumab, bivalirudin, bornauzumab, botulinum toxin type a, botulinum toxin type B, brentuximab, broudluumab, buserelin, C1 esterase inhibitor (human), cl esterase inhibitor (recombinant), canakinumab, carpuma, certolizumab, cetuximab, chorionic gonadotropin alpha, chorionic gonadotropin (human), chorionic gonadotropin (recombinant), coagulation factor IX, coagulation factor VIIa, human coagulation factor X, coagulation factor a-subunit (recombinant), collagenase, alfacalcita, corticotropin, daclizumab, daptomycin, daratumumab, darbepoetin alpha, defibrotide, dinesleukin, dinolizumab, dessicin, digoxin immune Fab (ovine), rituximab, alfa streptokinase, tegaserod alpha, dolaglutide, eculizumab, efavirenzab, eflomustine alpha, eloxat sulfatase alpha, elozumab, enfuvirtide, epoetin alpha, epoetin delta, eptib, etanercept, elotuzumab, exenatide, factor IX complex (human), fibrinogen concentrate (human), fibrinolysin (plasmin), filgratin, filgrastim-sndz, follitropin alpha, follitropin beta, sulfatase, intragastric factor, gemtuzumab ozotain, glatiramer acetate, recombinant glucagon, glucosidase, rabies, brevibacterium peptide D, hepatitis a vaccine, hepatitis b virus, calcitonin, clostridium tetani, human blepharaonis, human serum albumin (human leukoplakia), human hyaluronidase, human serum albumin (human serum albumin), human serum albumin (human immunoglobulin D), hyaluronidase, hyaluronidase (human recombinant), ibuzumab, ibritumomab tiuxetan, idazumab, iduronate, imiglucerase, human immunoglobulin, infliximab, insulin aspart, insulin, gemtuzumab ozogamicin, glatiramer acetate, recombinant glucagon, glucosidase (glucarpiadase), golimumab, gramicidin D, hepatitis A vaccine, hepatitis B immunoglobulin, human calcitonin, human clostridium tetani toxoid immunoglobulin, human rabies virus immunoglobulin, <xnotran> Rho (D) , , - , , ( ), , , , , , , , , , , , , , , , , (), - (Insulin-isophane), α -2a , α -2b, alfacon-1, α -nl, α -n3, β -la, β -lb, γ -lb, , , , , , , , , , , , , , - β, , , , α , , , , , , , , , , ospA , , , , , , , , , , α -2a, α -2b, </xnotran> Pegylcol interferon beta-1 a, pegololase, pegvisomant, pembrolizumab, pertuzumab, swine lung phospholipid (Poractant alfa), pramlintide, preotact, protamine sulfate, human protein S, prothrombin complex concentrate, ragweed pollen extract, ramucirumab, ranibizumab, labrassin, ranibixin, reteplase, rilonacept, rituximab, romidepsin, glycosidase, salmon calcitonin, sargrastim, sartusin, sartomodulcimab pentosan, sebelipase alfa, secretogenesin, secukinumab, sertraline, albumin, iodinated serum albumin, tuximab, semamectin alpha, sipuleucel-T, growth hormone recombinant, streptokinase, sulodet, sulodexide, albumin recombinant recombinant proteins suxolectin α, taliosidase α, teduglutide, teicoplanin, tenecteplase, teriparatide, terxamine, thrombomodulin alpha, thymosin, thyroglobulin, thyrotropin α, tositumomab, trastuzumab, tuberculin purified protein derivatives, tolosin α, urofollitropin, urokinase, ustrocumab, vasopressin, vedolizumab, vealasidase α, thrombomodulin α, thymine, thyroglobulin, thyroid hormone, tosituzumab, tositumumab, trastuzumab, tuberculin purified protein derivatives, tolosin α, urofollitropin, urokinase, ustrocumab, vasopressin, vedolizumab, vedolastase α.
In another embodiment, the protein is selected from the group consisting of those included in Raghava, gajendra p.s.; usmani, salman Sadullah; bedi, gursimura; samuel, jesse s.; singh, sandep; kalra, sourav et al (2017): THPdb: database of FDA Approved Peptide and Protein Therapeutics, the contents of which are incorporated herein by reference.
In another embodiment, the cargo moiety is a pharmacologically active compound, a small molecule, a liposome encapsulating protein, a radionuclide or radionuclide-labeled compound, a nucleic acid (e.g., siRNA, shRNA, miRNA, phosphorothioate-modified RNA, an aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof). For example, a labeled binding protein of the invention can be derived by functionally linking an antibody or antibody portion of the invention (by chemical coupling, genetic fusion, non-covalent binding, or otherwise) to one or more other molecular entities (e.g., another antibody (e.g., a bispecific antibody or diabody), a detectable agent, an agent, a protein or peptide that can mediate the binding of the antibody or antibody portion to another molecule (e.g., a streptavidin core region or a polyhistidine tag), and/or a cytotoxic or therapeutic agent selected from the group consisting of mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors of gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, anti-hormonal agents, corticosteroids, photosensitizing agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, radiosensitizing agents, and combinations thereof).
The binding proteins of the invention may be immobilized on a solid support. Solid supports are known in the art and include, for example, plates, beads or chromatography resins, for example, trisacryl, sepharose (sepharose), agarose (agarose), polyacrylamide, poros, poroshell, captol, toyopearl, hypercel, cellulose-type, sephadex (dextran), amberlite, amberchrome, amberjet, dowex, optipore, subpak, combigel, monosphere, duolite, diaion, aminolink, sulfolink, carboxylink, glycoink, marathon or glycoattach. In one embodiment, the bead or chromatography resin comprises protein a sepharose or protein G sepharose.
In one embodiment, the antibody is conjugated to an imaging agent or a detection molecule or label (used interchangeably herein). Examples of imaging agents that can be used in the compositions and methods described herein include, but are not limited to, radioisotope labels (e.g., indium), enzymes (e.g., horseradish peroxidase), rayon light labels, luminescent labels, bioluminescent labels, magnetic labels, biotin, SULFO-TAG labeled streptavidin, alkaline phosphatase, cresyl violet, quantum dots, thioisothiocyanate rayon light (FITC), infrared molecules, or Electron Paramagnetic Resonance (EPR) spin tracer labels.
Detectable reagents useful for derivatizing antibodies or antibody moieties of the invention include fluorescent compounds. Exemplary entangling detectable agents include entangling light, isothiocyanate entangling light, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like.
Antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, and the like. When the antibody is derivatized with a detectable enzyme, detection is performed by adding an additional reagent that the enzyme uses to produce a detectable reaction product. For example, the addition of hydrogen peroxide and diaminobenzidine produces a detectable colored reaction product when the detectable reagent horseradish peroxidase is present. Antibodies can also be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding.
In another embodiment, the glycosylation of the antibody or antigen binding portion of the invention is modified. For example, aglycosylated antibodies can be made (i.e., antibodies lack glycosylation). Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such sugar modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region glycosylation sites, thereby eliminating glycosylation at that site. This aglycosylation may increase the affinity of the antibody for the antigen. Such methods are described in more detail in PCT publication WO2003016466A2 and U.S. patent nos. 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.
Additionally or alternatively, modified antibodies of the invention with altered glycosylation patterns can be prepared, such as low fucosylated antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNAc structures. This altered glycosylation pattern has been shown to increase the ADCC capacity of the antibody. Such sugar modification can be accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies of the invention to produce antibodies with altered glycosylation. See, e.g., shields, r.l. et al (2002) j.biol.chem.277:26733-26740; umana et al (1999) nat. Biotech.17:176-1 and European patent No. EP 1,176,195; PCT publication WO 03/035835; WO 99/54342, each of which is incorporated herein by reference in its entirety.
Protein glycosylation depends on the amino acid sequence of the protein of interest and the host cell expressing the protein. Different organisms can produce different glycosylases (e.g., glycosyltransferases and glycosidases) and have different available substrates (nucleotide sugars). Due to these factors, the glycosylation pattern of the protein and the composition of glycosyl residues may differ (depending on the host system expressing the particular protein). Glycosyl residues useful in the present invention can include, but are not limited to, glucose, galactose, mannose, fucose, N-acetylglucosamine, and sialic acid. Preferably, the glycosylation binding protein contains glycosyl residues, so that the glycosylation pattern is human.
It is known to the person skilled in the art that different glycosylation of proteins can lead to different protein characteristics. For example, a therapeutic protein produced in a microbial host (e.g., yeast) and glycosylated using the yeast endogenous pathway may have reduced potency as compared to the same protein expressed in a mammalian cell (e.g., a CHO cell line). Such glycoproteins may also be immunogenic in humans and exhibit a reduced in vivo half-life after administration. Specific receptors in humans and other animals recognize specific glycosyl residues and promote rapid clearance of proteins from the bloodstream. Other side effects may include changes in protein folding, solubility, sensitivity to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or sensitization. Thus, the practitioner may prefer a therapeutic protein having a particular composition and glycosylation pattern, e.g., a glycosylation composition and pattern that is the same as or at least similar to that produced in human cells or species-specific cells of the subject animal of interest.
Expression of a glycosylated protein that is different from the host cell may be achieved by genetically modifying the host cell to express a heterologous glycosylase. Using techniques known in the art, one can generate antibodies or antigen-binding portions thereof that exhibit glycosylation of human proteins. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylases such that the glycosylated proteins (glycoproteins) produced in these yeast strains exhibit the same protein glycosylation as animal cells, particularly human cells (U.S. patent publication nos. 20040018590 and 20020137134 and PCT publication No. WO2005100584 A2).
In addition to binding proteins, the invention also relates to anti-idiotype (anti-Id) antibodies specific for such binding proteins of the invention. An anti-Id antibody is an antibody that recognizes a unique determinant that is normally associated with the antigen binding region of another antibody. anti-Id can be prepared by immunizing an animal with a binding protein or CDR-containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody. anti-Id antibodies can also be used as an "immunogen" to induce an immune response in another animal, thereby generating what is known as an anti-Id antibody.
Furthermore, one skilled in the art will appreciate that libraries of host cells genetically engineered to express various glycosylation enzymes can be used to express a protein of interest such that member host cells of the library produce a protein of interest with different glycosylation patterns. The practitioner can then select and isolate a protein of interest with a particular novel glycosylation pattern. Preferably, proteins with a particular selected novel glycosylation pattern exhibit improved or altered biological properties.
Antibodies can be produced by any of a variety of techniques. For example, by host cell expression, wherein the expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" of various forms is intended to include usually used to introduce exogenous DNA into prokaryotic or eukaryotic host cells in various techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. Although expression of the antibody in prokaryotic or eukaryotic host cells is possible, it is preferred to express the antibody in eukaryotic cells, most preferably mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete an antibody of appropriate folding and immunological activity.
Preferred mammalian host cells for expression of the recombinant antibodies disclosed herein include chinese hamster ovary (CHO cells) (including DHFR-CHO cells, which are described in Urlaub and Chasin, (1980) proc.natl.acad.sci.usa 77, 4216-4220, used with DHFR selection markers, e.g., as described in r.j.kaufman and p.a.sharp (1982) mol.biol.159: 601-621), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow the antibody to be expressed in the host cell, or more preferably, to secrete the antibody into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.
Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of a light and/or heavy chain of an antibody of the invention. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that is not essential for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the present disclosure. In addition, crosslinking an antibody of the disclosure with a second antibody by standard chemical crosslinking methods can produce a bifunctional antibody, wherein one heavy chain and one light chain are antibodies of the disclosure, and the other heavy chain and light chain are specific for an antigen other than the antigen of interest.
In a preferred system for recombinant expression of the antibody, or antigen-binding portion thereof, recombinant expression vectors encoding the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows for the use of methotrexate selection/amplification to select CHO cells that have been transfected with the vector. The selected transformant host cells are cultured to allow expression of the heavy and light chains of the antibody, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture the host cells, and recover the antibodies from the culture medium. Still further, the present disclosure provides methods of synthesizing recombinant antibodies by culturing the host cell in a suitable medium until the recombinant antibody is synthesized. Recombinant antibodies can be produced using nucleic acid molecules corresponding to the amino acid sequences disclosed herein. In one embodiment, the nucleic acid molecules shown in SEQ ID NOS: 25-45 (see Table 2 below) are used to produce recombinant antibodies. The method may further comprise isolating the recombinant antibody from the culture medium.
B. Method for preparing anti-TAT peptide antibody
Antibodies of the invention can be prepared by any of a variety of techniques known in the art.
General methods for preparing monoclonal antibodies by hybridomas are well known. Immortal antibody-producing cell lines can also be generated by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with EB virus. See, e.g., m.schreier et al, "hybrid Techniques" (1980); hammering et al, "Monoclonal Antibodies And T cell hybrids" (1981); kennett et al, "Monoclonal Antibodies" (1980); see also, U.S. patent nos. 4,341,761;4,399,121;4,427,783;4,444,887;4,451,570;4,466,917;4,472,500;4,491,632 and 4,493,890. Methods for producing polyclonal antibodies are well known in the art. See, U.S. Pat. No. 4,493,795 to Nestor et al.
A panel of monoclonal antibodies raised against TAT peptides (e.g., TAT transduction domains) can be screened for by various properties (i.e., subtype, epitope, affinity, etc.).
Monoclonal antibodies, typically comprising Fab and/or F (ab') of the useful antibody molecule 2 In part, can be edited using Antibodies-A Laboratory Manual, harlow and Lane, coPrepared by the hybridoma technique described in ld Spring Harbor Laboratory, new York (1988), which is incorporated herein by reference. Briefly, to form hybridomas that produce monoclonal antibody compositions, a myeloma or other self-sustaining cell line is fused to lymphocytes obtained from the spleen of a mammal hyperimmunized with an appropriate TAT peptide (e.g., a TAT transduction domain).
Polyethylene glycol (PEG) 6000 is commonly used to fuse spleen cells with myeloma cells. Fusion hybrids are selected by their sensitivity to HAT. Hybridomas that produce monoclonal antibodies useful in the practice of the present invention are identified by their ability to immunoreact with an antibody or binding member of the present invention and their ability to inhibit specific tumorigenic or hyperproliferative activities in target cells.
Monoclonal antibodies useful in the practice of the invention can be produced by initiating culture of monoclonal hybridomas comprising a nutrient medium comprising hybridomas that secrete antibody molecules with the appropriate antigen specificity. The culture is maintained for a period of time and under conditions sufficient for the hybridoma to secrete the antibody molecule into the culture medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques.
Media useful for preparing these compositions are well known in the art and are commercially available, and include synthetic media, inbred mice, and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; dulbecco et al, virol.8:396 (1959)) supplemented with 4.5gm/l glucose, 20mm glutamine and 20% fetal bovine serum. An exemplary inbred mouse strain is Balb/c.
1. Preparation of anti-TAT peptide monoclonal antibodies using hybridoma technology
Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant, and phage display techniques, or a combination thereof. For example, monoclonal Antibodies can be generated using hybridoma technology, including those known in the art and described, for example, in Harlow et al, antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed.1988); hammerling et al, in: monoclone Antibodies and T-Cell hybrids 563-681 (Elsevier, N.Y., 1981), the entire contents of which are incorporated herein by reference. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) rather than the method by which it was produced.
Methods for generating and screening specific antibodies using hybridoma technology are routine and well known in the art. For example, monoclonal antibodies can be produced by culturing hybridoma cells that secrete an antibody of the invention, wherein, preferably, the hybridomas are produced by fusing spleen cells isolated from a mouse immunized with an antigen of the invention with myeloma cells, and then screening the hybridomas produced by the fusion to obtain hybridoma clones that secrete antibodies that bind a polypeptide of the invention. Briefly, mice can be immunized with a TAT peptide antigen (e.g., a TAT transduction domain antigen). In certain embodiments, a TAT peptide antigen (e.g., a TAT transduction domain antigen) is administered with an adjuvant to stimulate an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immune stimulating complex). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a localized deposit, or they may comprise substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if the polypeptide is administered, the immunization regimen will comprise administering the polypeptide two or more times over a period of several weeks.
After immunizing an animal with the TAT peptide antigen, antibodies and/or antibody-producing cells may be obtained from the animal. Serum containing anti-TAT peptide antibodies was obtained from animals by blood sampling or by sacrifice. Serum obtained from the animal may be used directly, immunoglobulin fractions may be obtained from the serum, or anti-TAT peptide antibodies may be purified from the serum. The sera or immunoglobulins obtained in this way are polyclonal and therefore have a range of properties of heterogeneity.
Once an immune response is detected, for example, antibodies specific for the antigen TAT peptide (e.g., TAT transduction domain antigen) are detected in the mouse serum, i.e., mouse spleens are harvested and splenocytes isolated. The spleen cells are then fused with any suitable myeloma cells (e.g., cells of cell line SP20, available from ATCC) by well-known techniques. Hybridomas were selected and cloned by limiting dilution. Cells secreting antibodies capable of binding to a TAT peptide (e.g., a TAT transduction domain) in the hybridoma clones are then assayed by methods known in the art. Ascites fluid, which typically contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.
In another embodiment, antibody-producing immortalized hybridomas can be prepared by immunizing an animal. Following immunization, the animals are sacrificed and splenic B cells are fused with immortal myeloma cells, as is well known in the art. See, e.g., harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). Following fusion and antibiotic selection, hybridomas are screened using TAT peptides or portions thereof or cells expressing TAT peptides or portions thereof. The initial screening was performed using enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA). An example of an ELISA screen is provided in WO00/37504, which is incorporated herein by reference.
Hybridomas producing anti-TAT peptide antibodies are selected, cloned, and further screened for desirable characteristics, including robust hybridoma growth, high antibody production, and desirable antibody characteristics, as discussed further below. Hybridomas can be cultured and expanded in vivo in syngeneic animals, in animals lacking the immune system (e.g., nude mice), or in vitro in cell culture. Methods of selecting, cloning and expanding hybridomas are well known to those skilled in the art.
In a preferred embodiment, the hybridoma is a mouse hybridoma, as described herein. In another preferred embodiment, the hybridoma is produced in a non-human, non-mouse species (e.g., rat, sheep, pig, goat, cow, or horse). In another embodiment, the hybridoma is a human hybridoma in which a human non-secretory myeloma is fused to human cells expressing an anti-TAT peptide antibody.
Antibody fragments recognizing specific epitopes can be prepared by known techniquesAnd (4) generating. For example, fab and F (ab') 2 Fragments may be generated by using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab') 2 Fragments)) are produced by proteolytic cleavage of the immunoglobulin molecule. F (ab') 2 Fragments comprise the variable region, the light chain constant region, and the CH1 domain of the heavy chain.
2. Production of anti-TAT peptide monoclonal antibodies using selective lymphocyte antibody method
In another aspect of the invention, recombinant antibodies are produced from individual isolated lymphocytes using a method known in the art as the Selected Lymphocyte Antibody Method (SLAM), as described in U.S. Pat. No. 5,627,052, PCT publication No. WO92/02551 and Babcock, J.S. et al, (1996) Proc. Natl. Acad. Sci. USA93: 7843-7848. In this method, antigen-specific hemolytic plaque assays are used to screen individual cells secreting an antibody of interest, such as lymphocytes derived from any of the above-mentioned immunized animals, wherein the antigen TAT peptide, a subunit of the TAT peptide (e.g., a TAT transduction domain antigen), or a fragment thereof, is coupled to sheep erythrocytes using a linker (e.g., biotin) and used to identify individual cells secreting an antibody specific for the TAT peptide (e.g., a TAT transduction domain). After identification of antibody-secreting target cells, the heavy and light chain variable region cdnas are rescued from the cells by reverse transcriptase-PCR and then expressed in mammalian host cells (e.g., COS or CHO cells) in the context of appropriate immunoglobulin constant regions (e.g., human constant regions). Host cells are transfected with amplified immunoglobulin sequences derived from lymphocytes selected in vivo, and then further in vitro analysis and selection can be performed, for example, by panning the transfected cells to isolate cells expressing antibodies to TAT peptides (e.g., TAT transduction domains). The amplified immunoglobulin sequences can be further manipulated in vitro, for example, by in vitro affinity maturation methods, such as those described in PCT publication Nos. WO97/29131 and WO 00/56772.
3. Generation of anti-TAT peptide monoclonal antibodies using recombinant antibody libraries
In vitro methods are also useful for preparationThe antibodies of the invention, wherein the antibody library is screened to identify antibodies with the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include, for example, ladner et al, U.S. patent nos. 5,223,409; kang et al, PCT publication No. WO 92/18619; dower et al, PCT publication No. WO 91/17271; winter et al, PCT publication No. WO 92/20791; markland et al, PCT publication No. WO 92/15679; breitling et al, PCT publication No. WO 93/01288; mcCafferty et al, PCT publication No. WO92/01047; garrrard et al, PCT publication No. WO 92/09690; fuchs et al (1991) Bio/Technology91370-1372; hay et al, (1992) Hum antibody hybrids 3; huse et al, (1989) Science 246, 1275-1281; mcCafferty et al, nature (1990) 348; griffiths et al, (1993) EMBO J12; hawkins et al, (1992) J Mol Biol 226; clackson et al, (1991) Nature 352; gram et al, (1992) PNAS 89; garrad et al, (1991) Bio/Technology 9; hoogenboom et al, (1991) Nuc Acid Res 19; and Barbas et al, (1991) PNAS 88, 7978-7982, methods described in us patent application publication No. 20030186374 and PCT publication No. WO97/29131, the contents of each of which are incorporated herein by reference.
The recombinant antibody library may be from a subject immunized with a TAT peptide or a portion of a TAT peptide (e.g., a TAT transduction domain). Alternatively, the recombinant antibody library may be from a natural subject, i.e., a subject not immunized with TAT peptide, such as a human antibody library from a human subject not immunized with TAT peptide. Antibodies of the invention are selected by screening a recombinant antibody library with peptides comprising TAT peptides, thereby selecting those antibodies that recognize TAT peptides (e.g., TAT transduction domains). Methods for performing such screening and selection are well known in the art, for example as described in the references in the preceding paragraphs. To select antibodies of the invention having a particular binding affinity for a TAT peptide (e.g., a TAT transduction domain), e.g., with a particular k off Those with rate constants that dissociate from TAT transduction domains, methods known in the art for surface plasmon resonance, can be used to select for compounds with a desired k off Antibody with rate constant. To select for specific neutralizing activity against TAT transduction DomainAntibodies of the invention, e.g. with specific IC 50 Can be used using standard methods known in the art for assessing inhibition of TAT peptide activity.
In one aspect, the invention relates to an isolated antibody or antigen-binding portion thereof that binds to a TAT peptide, particularly a human TAT peptide. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.
For example, the antibodies of the invention can also be produced using various phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. In particular, such phage can be used to display antigen binding domains expressed by repertoires (retetors) or combinatorial antibody libraries (e.g., human or murine). Phage expressing an antigen binding domain that binds an antigen of interest can be selected or identified with the antigen (e.g., using a labeled antigen or an antigen bound or captured on a solid surface or bead). The phage used in these methods are typically filamentous phage comprising fd and M13 binding domains expressed by the phage, in which Fab, fv or disulfide stabilized Fv antibody domains are recombinantly fused to phage gene III or gene VIII proteins. Examples of phage display methods that can be used to prepare antibodies of the invention include Brinkman et al, j.immunol.methods 182; ames et al, j.immunol.methods 184 (1995); kettleborough et al, eur.J. Immunol.24:952-958 (1994); persic et al, gene 187-18 (1997); burton et al, advances in Immunology 57 (1994); PCT application No. PCT/GB91/01134; PCT publication No. WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. patent nos. 698,426;5,223,409;5,403,484;5,580,717;5,427,908;5,750,753;5,821,047;5,571,698;5,427,908;5,516,637;5,780,225;5,658,727;5,733,743, and 5,969,108, each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, antibody coding regions can be isolated from the phage and used to produce antibodies, including human antibodies, or any of themThe whole antibody of his desired antigen binding fragment and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, fab 'and F (ab') can also be produced recombinantly using methods known in the art 2 Fragment techniques, such as PCT publication WO92/22324; mullinax et al, bioTechniques12 (6): 864-869 (1992); and Sawai et al, AJRI 34 (1995); and Better et al, science 240, 1041-1043 (1988) (which references are incorporated herein by reference in their entirety). Examples of techniques that can be used to produce single chain Fv's and antibodies include U.S. Pat. nos. 4,946,778 and 5,258,498; huston et al, methods in Enzymology203:46-88 (1991); shu et al, PNAS 90; and those described in Skerra et al, science 240-1040 (1988).
As an alternative to screening recombinant antibody libraries by phage display, other methods known in the art for screening large combinatorial libraries can be used to identify the dual specific antibodies of the invention. One type of alternative expression system is one in which a library of recombinant antibodies is expressed as RNA-protein fusions, as described in PCT publication No. WO98/31700 to Szostak and Roberts, and in Roberts, R.W., and Szostak, J.W. (1997) Proc.Natl.Acad.Sci.USA 94. In this system, a covalent fusion is made between the mRNA and its encoded peptide or protein by in vitro translation of a synthetic mRNA carrying puromycin (a peptidyl receptor antibiotic) at its 3' end. Thus, specific mrnas may be enriched from a complex mixture of mrnas (e.g., a combinatorial library) based on the properties of the encoded peptide or protein (e.g., an antibody or portion thereof), such as binding of the antibody or portion thereof to a dual specific antigen. The nucleic acid sequences encoding the antibodies or portions thereof recovered from the screening of these libraries may be expressed by recombinant means (e.g., in mammalian host cells) as described above, and in addition, further affinity maturation may be performed by additional rounds of screening for mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence, or by other methods for in vitro affinity maturation of recombinant antibodies as described above.
In another approach, the antibodies of the invention can also be produced using yeast display methods known in the art. In the yeast display method, genetic methods can be used to link the antibody domain to the yeast cell wall and display it on the yeast surface. In particular, such yeast can be used to display the expression of all components or combinatorial antibody libraries (e.g., human or murine) antigen binding domains. Examples of yeast display methods that can be used to prepare the antibodies of the invention include those disclosed by Wittrup et al (U.S. Pat. No. 6,699,658), which is incorporated herein by reference.
4. Recombinant anti-TAT peptide antibodies
Antibodies of the invention can be produced by any of a variety of techniques known in the art. For example, by host cell expression, wherein the expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" of various forms is intended to include usually used to introduce exogenous DNA into prokaryotic or eukaryotic host cells in various techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. Although it is possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, most preferably mammalian host cells, since such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete an antibody of appropriate folding and immunological activity.
In certain embodiments, the invention features an isolated nucleic acid encoding a binding protein amino acid sequence described herein. In other certain embodiments, the invention also features isolated nucleic acids encoding the antibody construct amino acid sequences described herein. In the method of production, the expression vector comprises an isolated nucleic acid. Non-limiting examples of such expression vectors are pUC series vectors (Fermentas Life Sciences), pBluescript series vectors (Stratagene, la Jolla, calif.), pET series vectors (Novagen, madison, wis.), pGEX series vectors (Pharmacia Biotech, uppsala, sweden), and pEX series vectors (Clontech, palo Alto, calif.).
The host cell comprises a vector as described herein. According to an embodiment of the invention, the host cell is a prokaryotic cell or a eukaryotic cell. For example, the prokaryotic host cell is an E.coli cell. Eukaryotic cells may be selected from protozoan cells, animal cells, plant cells or fungal cells. The animal cells may be selected from mammalian cells, avian cells and insect cells. Preferably, the host cell is selected from the group consisting of CHO cells, COS cells, yeast cells and insect Sf9 cells. In a further related embodiment, the yeast cell is a Saccharomyces cerevisiae cell.
Preferred mammalian host cells for expression of recombinant antibodies of the invention include chinese hamster ovary (CHO cells) (including DHFR-CHO cells, which are described in Urlaub and Chasin, (1980) proc.natl.acad.sci.usa 77, 4216-4220, together with DHFR selection markers, e.g., as described in r.j.kaufman and p.a.sharp (1982) mol.biol.159: 601-621), NS0 myeloma, COS and SP2 cells. When a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow the antibody to be expressed in the host cell, or more preferably, to secrete the antibody into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.
Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedure are within the scope of the invention. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of a light and/or heavy chain of an antibody of the invention. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that is not essential for binding to the antigen of interest. Molecules expressed by such truncated DNA molecules are also included in the antibodies of the invention. In addition, crosslinking an antibody of the invention with a second antibody by standard chemical crosslinking methods can produce a bifunctional antibody in which one heavy chain and one light chain is an antibody of the invention and the other heavy and light chains are specific for an antigen other than the antigen of interest.
In a preferred system for recombinant expression of the antibodies or antigen-binding portions thereof of the invention, recombinant expression vectors encoding the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows for the use of methotrexate selection/amplification to select CHO cells that have been transfected with the vector. The selected transformant host cells are cultured to allow expression of the heavy and light chains of the antibody, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture the host cells, and recover the antibodies from the culture medium. The invention also provides methods for synthesizing a recombinant antibody of the invention by culturing a host cell of the invention in a suitable culture medium until the recombinant antibody of the invention is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.
The invention also relates to various methods of producing a protein capable of binding to TAT peptide, producing an antibody or antigen-binding portion thereof that binds to TAT peptide, said method comprising culturing a host cell as described herein in a culture medium, thereby expressing the nucleic acid and producing the antibody. An exemplary method of producing a protein capable of binding to a TAT peptide comprises culturing a host cell as described herein in a culture medium under conditions sufficient to produce a binding protein capable of binding to a TAT peptide.
The invention also features proteins produced according to the methods.
5. Humanized anti-TAT peptide antibodies
Humanized antibodies are antibody molecules from non-human species antibodies that bind the desired antigen, having one or more Complementarity Determining Regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Known human Ig sequences are disclosed, for example, in the following: nbi.nih.gov/entrez-/query.fcgi; atcc.org/phase/hdb.html; com/; com/; (xiv) antibodyresource. Com/onlinecomp. Html; public, state, edu/. About, predo/research _ tools, html; un.uni-heidelberg.de/SD/IT/IT.html; com/immunology/CH-05/kuby05.Htm; library. Thinkquest. Org/12429/Immune/antibody. Html; org/gradts/lectures/1996/vlab/; path, cam, ac, uk/. About, mrc 7/m-ikeimagees. Html; com/; mcb. Harvard. Edu/BioLinks/Immuno-logy, imimenologylink. Com/; pathbox.wustl.edu/. About.hcenter/index "-html; biotech. Ufl. Edu/. About. Hcl/; com/pa/340913. Html-; nal. Usda. Gov/awic/pubs/antibody/; m.ehime-u.acjp/. About.yasuhito-/elisa.html; com/table. Asp; html icnet, uk/axp/facs/davies/lines-ks; biotech. Ufl.edu/. About. Fccl/protocol. Html; (iii) isac-net.org/sites _ geo.html; imt. Uni-marburg. De/. About. Rek/AEP-Start. Html; basev.uci.kun.nl/. About.plates/links.html; una-hd, de/immuno, bme, nwu, edu/; src-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html; ibt. Unam. Mx/vir/V _ mic. Html; imgt.cnusc.fr 8104/; html, biochem. Ucl. Ac. Uk/. About. Martin/abs/index. Html; anti.bath.ac.uk/; abgen. Cvm. Tamu. Edu/lab/wwwabgen. Html; uniz.ch/. About.honegger/AHOsem-inar/Slide01.Html; cryst.bbk.ac.uk/. About.about.ubcg07s/; nmr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; path, cam.ac.uk/. About.mrc7/h-umanisation/TAHHP.html; html/vir/structure/stat _ aim.html; html. Missouri. Edu/smithgp/index. Html; cryst, bioc, cam, ac, uk/. Abo-ut, fmolina/Web-pages/Pept/spottech, html; jerni.de/frroducts.htm; com/ibm.html., kabat et al, sequences of Proteins of Immunological Interest, u.s.dept.health (1983), each of which is incorporated herein by reference in its entirety. Such introduced sequences may be used to reduce immunogenicity or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life or any other suitable characteristic known in the art.
Framework residues in the human framework regions may be substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction of the CDRs and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify framework residues that are unique at a particular position. (see, e.g., queen et al, U.S. Pat. No. 5,585,089; riechmann et al, nature 332 (1988), which is incorporated herein by reference in its entirety). Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that elucidate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These displayed checks allow analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and input sequences to achieve desired antibody characteristics, such as enhanced affinity for the target antigen. Generally, CDR residues are directly and most essentially involved in influencing antigen binding. Antibodies can be humanized using a variety of techniques known in the art, such as, but not limited to, jones et al, nature 321 522 (1986); verhoeyen et al, science 239 1534 (1988)); sims et al, J.Immunol.151:2296 (1993); chothia and Lesk, J.mol.biol.196:901 (1987); carter et al, proc. Natl. Acad. Sci. U.S. A.89:4285 (1992); presta et al, J.Immunol.151:2623 (1993), padlan, molecular Immunology 28 (4/5): 489-498 (1991); studnicka et al, protein Engineering 7 (6): 805-814 (1994); roguska, et al, PNAS 91; PCT publication No. WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5814476, 5763192, 5723323, 5,766886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539;4,816,567, each of which is incorporated herein by reference, and including references cited therein.
The invention encompasses humanized anti-TAT peptide antibodies.
Use of anti-TAT peptide binding proteins
A. Detection of TAT fusion molecules comprising TAT peptides
Given their ability to bind to TAT protein transduction domains, anti-TAT binding proteins of the invention (e.g., antibodies or portions thereof) may be used to detect TAT (e.g., in a biological sample, such as serum, plasma, urine, tissue, or cells) using conventional immunoassays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), antibody-labeled fluorescence imaging, or tissue immunohistochemistry. It is to be understood that the present invention encompasses the detection of any fragment of a TAT peptide polypeptide, provided that the fragment allows the specific identification of the TAT peptide by the detection method of the present invention. In a particular embodiment, a TAT peptide binding protein of the invention specifically binds to a TAT protein transduction domain. For example, an ELISA antibody must be able to bind to a TAT peptide fragment (e.g., a TAT protein transduction domain) to enable detection.
In another embodiment, TAT binding proteins (e.g., antibodies) of the invention can be used to detect a transcellular delivery system, such as TAT-liposomes or TAT-nanoparticles, for example, for delivery of small molecules.
In one embodiment, a TAT binding protein (e.g., an antibody) of the invention can be used to detect a TAT peptide as part of an intact TAT fusion molecule. For example, TAT binding proteins of the invention can be used to detect intact TAT fusion molecules comprising a TAT protein transduction domain linked to a cargo moiety. In one embodiment, a TAT binding protein (e.g., an antibody) specifically binds to a TAT peptide, e.g., a TAT protein transduction domain, of an intact TAT fusion molecule and does not bind to the cargo portion of the TAT fusion molecule.
In one embodiment, the cargo moiety is a polypeptide. In one embodiment, the polypeptide is an ataxin, e.g., the TAT fusion molecule is a TAT-ataxin fusion molecule. In other embodiments, the cargo moiety is a nucleic acid molecule, such as DNA, mRNA, siRNA, shRNA.
In some embodiments, TAT binding proteins (e.g., antibodies) of the invention can be used to detect, measure, validate the delivery of, or quantify TAT peptides (e.g., TAT fusion molecules comprising a TAT protein transduction domain) in vitro or in vivo. Thus, a binding protein of the invention is capable of detecting, quantifying, validating the delivery, and/or measuring a cargo moiety (e.g., a prodrug or drug) coupled to a TAT peptide (e.g., a TAT fusion molecule comprising a TAT protein transduction domain) in vivo or in vitro (e.g., in a particular tissue).
In some embodiments, TAT binding proteins (e.g., antibodies) of the invention may be used to image tissue, for example, by perfusing the tissue with a labeled anti-TAT binding protein. In some embodiments, TAT binding proteins (e.g., antibodies) of the invention can be used to detect, quantify, and monitor or track the pharmacokinetics, delivery, and/or localization of TAT peptides (e.g., TAT fusion molecules comprising a TAT protein transduction domain) by labeling the antibodies using, for example, infrared conjugates, radioisotope labels, or Electron Paramagnetic Resonance (EPR) spin tracer labels as described herein.
In one aspect, the methods of the invention provide methods for detecting and/or quantifying the level of a TAT fusion molecule in a sample (e.g., a biological sample) comprising contacting the sample with a binding protein of the invention under conditions such that the binding protein binds to a TAT protein transduction domain in the sample, thereby detecting and/or quantifying the level of the TAT fusion molecule in the sample. In one embodiment, the biological sample is a liquid (e.g., blood) sample or a tissue sample.
In another aspect, the methods of the invention provide a method of detecting and/or quantifying the level of a TAT fusion molecule in vivo, comprising contacting a sample with a binding protein of the invention and imaging the binding protein, under conditions such that the binding protein binds to the TAT protein transduction domain, thereby detecting and/or quantifying the level of the TAT fusion molecule in vivo.
In some embodiments, the TAT fusion molecule comprises a TAT protein transduction domain covalently linked to a cargo moiety. In some embodiments, the cargo moiety is a polypeptide. In some embodiments, the cargo moiety is an ataxin polypeptide.
In another embodiment, the cargo moiety is a pharmacologically active compound, a small molecule, a liposome encapsulating protein, a radionuclide or radionuclide-labeled compound, a nucleic acid (e.g., siRNA, shRNA, miRNA, phosphorothioate-modified RNA, an aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof).
In some embodiments, the methods of the invention further comprise assessing the stability of the TAT fusion molecule using the binding protein of the invention by, for example, detecting the TAT fusion molecule described herein and measuring the stability of the TAT fusion molecule over time.
In another aspect, the methods of the invention provide methods for isolating and/or purifying a TAT fusion molecule present in a mixture, wherein the TAT fusion molecule comprises a TAT protein transduction domain covalently attached to a cargo moiety, comprising (a) contacting a mixture comprising the TAT fusion molecule with an immobilized binding protein of the invention under conditions such that the TAT fusion molecule binds to the immobilized binding protein; (b) eluting the TAT fusion molecule from the immobilized binding protein.
In some embodiments, the binding proteins of the invention can be used to enrich for intact TAT fusion molecules in a mixture. For example, the mixture may comprise TAT fusion molecules in intact and degraded forms.
In some embodiments, the binding protein of the invention is labeled with horseradish peroxidase, ruthenium terpyridyl, alkaline phosphatase, cresyl violet, quantum dots, fluorescein Isothiocyanate (FITC), infrared molecules, radioisotope labels, or EPR spin tracer labels.
In one embodiment, an ELISA assay is used to detect and/or quantify TAT peptides comprising a TAT protein transduction domain or TAT fusion molecules comprising a TAT protein transduction domain. In an exemplary ELISA, a binding protein (e.g., an antibody that binds to a TAT protein transduction domain) is immobilized on a selected surface that exhibits protein affinity, e.g., on wells in a polystyrene microwell plate. A test composition or sample, such as a blood or tissue sample containing a TAT protein transduction domain (e.g., a TAT protein transduction domain fused to a cargo moiety), is then added to the well. After binding and washing to remove non-specifically bound immune complexes, bound antigen can be detected. Detection is typically achieved by the addition of a second antibody specific for the target protein linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody followed by the addition of a third antibody having binding affinity for the second antibody, wherein the third antibody is linked to a detectable label.
In another exemplary ELISA, a sample that may contain a TAT protein transduction domain (e.g., a TAT fusion molecule comprising a TAT protein transduction domain) is immobilized on the surface of a well and then contacted with an anti-TAT peptide antibody of the invention. Bound antigen is detected after binding and washing to remove non-specifically bound immune complexes. When the initial antibody is linked to a detectable label, the immune complex can be detected directly. Likewise, the immune complex can be detected using a second antibody having binding affinity for the first antibody, wherein the second antibody is linked to a detectable label.
Whatever format is used, the ELISA has certain common features, such as coating, incubation or binding, washing to remove non-specifically bound material, and detection of bound immune complexes. These are described below.
Where the plate is coated with antigen or antibody, the wells of the plate are typically incubated with a solution of the antigen or antibody overnight or for a specified time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surface of the wells is then "coated" with a non-specific protein that is antigenically neutral to the test antisera. These include Bovine Serum Albumin (BSA), casein and milk powder solutions. The coating allows to block non-specific adsorption sites on the immobilization surface and thereby reduce the background caused by non-specific binding of antisera on the surface.
In ELISA, it may be more common to use a secondary or tertiary detection format rather than a direct detection procedure. Thus, after binding of the protein or antibody to the well, coating with a non-reactive material to reduce background and washing to remove unbound material, the immobilization surface is contacted with a control clinical or biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immunocomplex then requires a labeled second binding ligand or antibody, or a second binding ligand or antibody that binds to a labeled third antibody or third binding ligand.
The phrase "under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include dilution of the antigen and antibody with a solution such as BSA, bovine Gamma Globulin (BGG), and Phosphate Buffered Saline (PBS)/Tween. These added reagents also help to reduce non-specific background.
"suitable" conditions also refer to incubation at a temperature and for a period of time sufficient to allow effective binding. The incubation step is typically carried out at a temperature of preferably about 25 to 27 ℃ for about 1 to 2 to 4 hours, or may be overnight at about 4 ℃.
After all incubation steps in the ELISA, the contacted surfaces were washed to remove uncomplexed material. Preferred washing procedures include washing with solutions (e.g., PBS/Tween or borate buffer). After the formation of specific immune complexes between the test sample and the initially bound substances, followed by washing, the presence of even minute amounts of immune complexes can be determined.
To provide a means of detection, the second or third antibody will be linked to an associated label to allow detection. Preferably, this will be an enzyme which will produce a colour development upon incubation with an appropriate chromogenic substrate. Thus, for example, it may be desirable to contact and incubate the first or second immunocomplex with a urease, glucose oxidase, alkaline phosphatase, or catalase-conjugated antibody for a period of time (e.g., 2 hours in a PBS-containing solution (e.g., PBS-Tween) at room temperature) under conditions that favor the development of further immunocomplex formation.
After incubation with the labeled antibody, subsequent washing to remove unbound material quantifies the amount of label, for example by incubation with chromogenic substrates (e.g., urea and bromocresol purple). Quantification is then achieved by measuring the degree of color development, for example using a visible spectrum spectrophotometer.
In certain embodiments, an alternative method of detecting TAT peptides using the anti-TAT peptide binding proteins of the invention is to use protein immunoprecipitation in conjunction with mass spectrometry (e.g., tandem mass spectrometry, such as multiple reaction monitoring mass spectrometry (IP-MRM)). IP-MRMs are known in the art and are described, for example, in Lin et al (Journal of protein Research,2013,12, 5996-6003).
B. Marking
The invention provides methods for detecting a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain) in a biological sample, the method comprising contacting the biological sample with a binding protein (e.g., an antibody or antibody portion) of the invention and detecting the binding protein (e.g., an antibody (or antibody portion)) that binds to the TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain), thereby detecting the TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain) in the biological sample. The binding protein is labeled directly or indirectly with a detectable substance to facilitate detection of bound or unbound antibody.
Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable entangling materials include umbelliferone, entangling light, isothiocyanato light, rhodamine, dichlorotriazinamine entangling light, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of suitable radioactive materials include 3 H、 14 C、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I、 177 Lu、 166 Ho or 153 Sm。
In some embodiments, the binding protein of the invention is labeled with horseradish peroxidase, SULFO-TAG labeled streptavidin, alkaline phosphatase, cresyl violet, quantum dots, or Fluorescein Isothiocyanate (FITC), infrared molecules, radioisotope labels, or Electron Paramagnetic Resonance (EPR) spin tracer labels.
One skilled in the art will recognize that a number of strategies can be used to label the binding proteins of the invention to enable their detection or discrimination in a mixture of particles. The label may be attached by any known method, including methods that utilize non-specific or specific interactions of the label and the target. The label may provide a detectable signal or affect the migration of the particle in an electric field. Furthermore, labeling can be accomplished directly or via a binding partner.
In some embodiments, the label comprises a binding partner that binds to a TAT peptide (e.g., such as a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain), such as a TAT peptide antibody described herein, wherein the binding partner is attached to a moiety that is fluorescent. The compositions and methods of the present invention can utilize a high-entangling portion (moiety) capable of emitting, for example, at least about 200 photons when simulated by emitting light by a laser at a portion's excitation wavelength, wherein said laser is focused on a spot containing said portion having a diameter of no less than about 5 microns, and wherein the total energy directed by said laser to said spot is no more than about 3 microjoules (microJoule). Portions of the compositions and methods suitable for use in the present invention are described in more detail below.
Entangling gloss markings include Near Infrared (NIR) entangling gloss, described, for example, in Frangioni,2003, current Opinions in Chemical biology,7 (5): 626, the contents of which are incorporated herein by reference.
In some embodiments, the invention provides a label for detecting a biomolecule comprising a binding partner of a biomolecule (e.g., a TAT peptide antibody as described herein) linked to a fluorophore moiety, wherein the fluorophore moiety is capable of emitting at least about 200 photons when mimicked by emission of light by a laser at a portion of an excitation wavelength, wherein the laser is focused on a spot containing the moiety that is no less than about 5 microns in diameter, and wherein a total energy directed by the laser to the spot is no more than about 3 microjoules. In some embodiments, a portion comprises a plurality of entangling light entities, e.g., about 2 to 4, 2 to 5,2 to 6,2 to 7, 2 to 8,2 to 9,2 to 10, or about 3 to 5,3 to 6,3 to 7,3 to 8,3 to 9, or 3 to 10 entangling light entities. In some embodiments, a portion comprises about 2 to 4 entangling light entities. The entangling light entity may be entangling light dye molecules. In some embodiments, the entanglement photo dye molecule comprises at least one substituted indole ring system wherein the substituent on the 3-carbon of the indole ring comprises a chemically reactive group or coupling species. In some embodiments, the dye molecule is an Alexa Fluor molecule selected from the group consisting of: alexa Fluor 488, alexa Fluor532, alexa Fluor647, alexa Fluor680, or Alexa Fluor 700. In some embodiments, the dye molecule is an Alexa Fluor molecule selected from the group consisting of: alexa Fluor 488, alexa Fluor532, alexa Fluor680, or Alexa Fluor 700. In some embodiments, the dye molecule is an Alexa Fluor647 dye molecule. In some embodiments, the dye molecules comprise a first type and a second type of dye molecules, e.g., two different Alexa Fluor molecules, e.g., wherein the first type and the second type of dye molecules have different emission spectra. The ratio of the number of dye molecules of the first type and the second type may be, for example, 4 to 1, 3 to 1, 2 to 1,1 to 2,1 to 3 or 1 to 4. The binding partner may be, for example, a TAT peptide antibody as described herein.
In some embodiments, the invention provides a label for detecting a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain), wherein the label comprises a binding partner for the TAT peptide and a fluorophore portion, wherein the fluorophore portion is capable of emitting at least about 200 photons when mimicked by emitting light by a laser at an excitation wavelength of the portion, wherein the laser is focused on a spot containing the portion having a diameter of no less than about 5 microns, and wherein a total energy directed to the spot by the laser is no more than about 3 microjoules. In some embodiments, the portion of light comprises a photon of light. In some embodiments, the light portion comprises a plurality of entangling light molecules, for example about 2 to 10, 2 to 8,2 to 6,2 to 4,3 to 10, 3 to 8, or 3 to 6 entangling light molecules. In some embodiments, the label portion comprises about 2 to 4 fluorescent molecules. In some embodiments, the entangled photo dye molecule comprises at least one substituted indole ring system wherein the substituent on the 3-carbon of the indole ring comprises a chemically reactive group or coupling species. In some embodiments, the entangling photonic molecule is selected from the group consisting of: alexa Fluor 488, alexa Fluor532, alexa Fluor647, alexa Fluor680, or Alexa Fluor 700. In some embodiments, the entangling photonic molecule is selected from the group consisting of: alexa Fluor 488, alexa Fluor532, alexa Fluor680, or Alexa Fluor 700. In some embodiments, the entangling light molecule is an Alexa Fluor647 molecule. In some embodiments, the binding partner comprises an anti-TAT peptide antibody as described herein.
Electron Paramagnetic Resonance (EPR) spin tracer labeling can also be used to detect TAT peptides, e.g., TAT protein transduction domains or TAT fusion molecules comprising TAT protein transduction domains, in which TAT antibodies are also labeled with an EPR tracer (see, e.g., hubbell et al, 1998, current Opinion in Structural biology,8 (5): 649, the contents of which are incorporated herein by reference).
As an alternative to labeling antibodies, TAT peptides (e.g., TAT protein transduction domains or TAT fusion molecules comprising TAT protein transduction domains) are determined in biological fluid samples by competitive immunoassay using TAT peptide standards labeled with a detectable substance and TAT peptide antibodies that are not labeled. In this assay, a biological sample, a labeled TAT peptide standard and a TAT peptide antibody are combined and the amount of labeled standard bound to unlabeled antibody is determined. The amount of TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain) in a biological sample is inversely proportional to the amount of labeled standard bound to the anti-TAT peptide antibody. Similarly, TAT peptides (e.g., TAT protein transduction domains or TAT fusion molecules comprising TAT protein transduction domains) are assayed in biological fluid samples by competitive immunoassays using TAT peptide standards labeled with a detectable substance and TAT peptide antibodies that are not labeled.
C. Therapeutic uses of the invention
The binding proteins (e.g., antibodies and antibody portions thereof) are preferably capable of neutralizing TAT activity in vivo and in vitro.
It has been previously found that TAT vaccination and administration of antibodies to TAT protein is protective against HIV-1 infection and can produce long-term inhibition. In addition, infusion of anti-TAT antibodies can also block extracellular TAT autocrine/paracrine transactivation of HIV-1 replication in the case of acute exposure (see, e.g., moreau et al, journal of General Virology (2004), 85,2893-2901 Bennaser, Y, et al, (2002) Virology 303,174-180 Ensoli, B, et al, (1990) Nature,345,84-86 Moreau, E, et al, (2004) J Virol 78,3792-3796 Re, M.C., furlini, G.; vignoli, M. (1995). J Acquir Immune Defic Syndr Hum Retrovirol 10,408-416, re, M.C., vignoli et al, (2001) J Clin Virol 21,81-89, richardson, M.W., mirchandani, J.et al, (2003). Biomed Pharmacother57,4-14 Tikhonov, I.et al, (2003) J Virol 77,3157-3166, steinaa, L.et al, (1994) Arch Virol 139,263-271, silvera, P.et al, (2002) J Virol 76,3800-3809, the contents of which are incorporated herein by reference.
Thus, the binding proteins of the invention can be used to inhibit TAT activity, for example in cell culture, in human subjects, or in other mammalian subjects, thereby blocking or inhibiting HIV infection. In one embodiment, the present disclosure provides a method for inhibiting TAT activity comprising contacting TAT with a binding protein (e.g., an antibody or antigen binding portion) such that TAT activity is inhibited.
In another embodiment, disclosed herein are methods for reducing TAT activity in a subject, advantageously in a subject having a TAT-related disease (e.g., HIV infection or AIDS). The present disclosure provides methods for reducing TAT activity in a subject having such a disease, the method comprising administering to the subject a binding protein, e.g., an antibody or antibody portion of the present disclosure, such that TAT peptide activity in the subject is reduced. Preferably, the subject is a human subject.
In another embodiment, disclosed herein is a method for inhibiting HIV-TAT protein activity in a subject, the method comprising administering to the subject an antigen binding protein of the invention, thereby inhibiting HIV-TAT protein activity in the subject.
Binding proteins (e.g., antibodies of the disclosure) can be administered to a human subject for therapeutic purposes. In addition, for veterinary purposes or as an animal model of human disease, a binding protein, such as an antibody of the present disclosure, can be administered to a non-human mammal comprising a TAT peptide capable of binding to the antibody. With respect to the latter, such animal models can be used to assess the therapeutic efficacy of the antibodies of the present disclosure (e.g., testing the dose and time course of administration).
Non-limiting examples of diseases that can be treated with binding proteins (e.g., antibodies and antigen-binding portions thereof) include HIV infection and AIDS and its associated symptoms.
In another aspect, the application features a method of treating (e.g., curing, suppressing, ameliorating, inhibiting, delaying or preventing the onset of, or preventing the recurrence or recurrence of) or preventing a TAT-related disorder in a subject. The method comprises the following steps: a binding protein, such as an anti-TAT peptide antibody or portion thereof described herein, is administered to a subject in an amount sufficient to treat or prevent a TAT-related disorder. A TAT antagonist (e.g., an anti-TAT antibody or portion thereof) may be administered to a subject alone or in combination with other treatment modalities described herein.
Binding proteins (e.g., antibodies and antigen-binding portions thereof) can be used alone or in combination to treat such diseases. It will be appreciated that the antibody or antigen-binding portion thereof can be used alone or in combination with other agents (e.g., therapeutic agents) selected by those skilled in the art for their intended purpose. For example, the additional agent may be a therapeutic agent recognized in the art as useful for treating a disease or disorder treated by the antibody (e.g., HIV or AIDS) or a symptom associated therewith. The additional agent may also be an agent that imparts a beneficial attribute to the therapeutic composition, such as an agent that affects the viscosity of the composition.
It should also be understood that the combinations included within the present disclosure are those that can be used for their intended purpose. The agents listed below are illustrative and not limiting. A combination that is part of the present disclosure may be an antibody of the present disclosure and at least one additional agent. A combination may also include more than one additional agent, such as two or three additional agents, if the combination is such that the resulting composition can perform its intended function.
Combination therapy may include one or more TAT antagonists (e.g., anti-TAT antibodies or portions thereof) formulated with and/or co-administered with one or more anti-HIV agents. Term(s) for"anti-HIV agent" means a drug for inhibiting or preventing HIV infection and AIDS, or treating or ameliorating the symptoms of HIV infection or AIDS. In one embodiment, the anti-TAT antibodies of the invention are administered in combination with one or more antiretroviral therapies (ART) including, but not limited to, non-nucleoside reverse transcriptase inhibitors (NNRTIs), such as efavirenz (Sustiva) TM ) Etravirine (Intelence) TM ) And nevirapine (Viramune) TM ) (ii) a Nucleoside or Nucleotide Reverse Transcriptase Inhibitors (NRTI), e.g. Abacavir TM (Ziagen TM ) And the combination emtricitabine/tenofovir (Truvada) TM )、Descovy TM (Tenofovir alafenamide/emtricitabine) and lamivudine-zidovudine (Combivir) TM ) (ii) a Protease Inhibitors (PI), e.g. atazanavir (Reyataz) TM ) Darunavir (Prezista) TM ) Fosamprenavir (Lexiva) TM ) And indinavir (Crixivan) TM ) (ii) a Entry or fusion inhibitors, e.g. enfuvirdi (Fuzeon) TM ) And maraviroc (Selzentry) TM ) (ii) a And integrase inhibitors, e.g. Letergevir (Isentress) TM ) And dolutegravir (tivecay) TM )。
In particular embodiments, the anti-TAT antibody or ADC may be administered alone or in combination with another anti-HIV agent that binds to or acts synergistically with the antibody to treat a disease associated with TAT activity.
Provided herein are methods for treating HIV or AIDS in a patient comprising administering to the patient an anti-TAT binding protein (e.g., an antibody or fragment thereof) of the invention, wherein the combination therapy exhibits a synergistic effect, e.g., a therapeutic synergistic effect, in said subject. As used herein, "synergistic" or "therapeutic synergy" refers to a phenomenon wherein treatment of a patient with a combination of therapeutic agents exhibits a therapeutically superior result to that achieved by the use of the individual components of the combination each at their optimal dosage. For example, a therapeutically superior result is a result in which the patient a) exhibits a lower incidence of adverse events while receiving therapeutic benefit equal to or greater than the therapeutic benefit of the individual components of the combination when each is administered as monotherapy at the same dose as in the combination, or b) does not exhibit dose limiting toxicity while receiving therapeutic benefit greater than the benefit that would be achieved if each is administered therapeutically at the same dose as in the combination.
The pharmaceutical composition may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antibody portion. A "therapeutically effective amount" is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody or antibody portion can be determined by one of skill in the art and will vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount wherein the therapeutically beneficial effect outweighs any toxic or detrimental effects of the antibody or antibody portion. By "prophylactically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in a subject prior to or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Dosage regimens can be adjusted to provide the best desired response (e.g., therapeutic or prophylactic response). For example, a single bolus dose may be administered, multiple doses may be administered over time, or the dose may be reduced or increased proportionally according to the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for a dosage unit form depends directly or indirectly on: (a) The unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) inherent limitations within the art of formulating such active compounds for use in individual therapeutic sensitivity.
An exemplary, non-limiting range of therapeutically or prophylactically effective amounts of the antibody or antibody portion is 0.1-20mg/kg, more preferably 1-10mg/kg. It should be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is also to be understood that for any particular subject, the specific dosing regimen should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
D. Diagnostic use of the invention
In some embodiments, any of the anti-TAT antibodies provided herein can be used to detect the presence of TAT and HIV in a biological sample. Detection includes quantitative or qualitative detection.
The binding proteins (e.g., antibodies) disclosed herein can be used for a variety of purposes, such as for detecting an HIV infection or diagnosing AIDS in a subject. These methods can include contacting a sample from a subject diagnosed with HIV or AIDS with a binding protein (e.g., an antibody) described herein, and detecting binding of the binding protein (e.g., an antibody) to the sample. An increase in binding of the binding protein (e.g., antibody) to the sample relative to binding of the binding protein (e.g., antibody) to the control sample confirms that the subject has HIV infection and/or AIDS. In some embodiments, the method further comprises contacting a second antibody that binds to the TAT peptide with the sample and detecting binding of the second antibody. In some non-limiting examples, an increase in binding of the antibody to the sample relative to a control sample detects TAT peptide in the subject, thereby diagnosing HIV infection in the subject.
According to another embodiment, the present invention provides a diagnostic method. Diagnostic methods generally include contacting a biological sample (e.g., blood, serum, saliva, urine, sputum, cell swab sample, or tissue biopsy sample) obtained from a patient with a TAT binding protein (e.g., an antibody) and determining whether the binding protein (e.g., antibody) preferentially binds to the sample as compared to a control sample or a predetermined cut-off value, thereby indicating the presence of the TAT peptide in the sample.
In some embodiments, anti-TAT binding proteins, e.g., antibodies, are provided for use in diagnostic or detection methods. In another aspect, a method of detecting the presence of a TAT peptide in a biological sample is provided. In some embodiments, a method comprises contacting a biological sample with an anti-TAT binding protein (e.g., an antibody) as described herein under conditions that allow binding of the anti-TAT binding protein (e.g., the antibody) to the TAT peptide, and detecting whether a complex is formed between the anti-TAT binding protein (e.g., the antibody) and the TAT peptide. Such methods may be in vitro or in vivo. In some embodiments, an anti-TAT binding protein (e.g., an antibody) is used to select a subject suitable for treatment with an anti-TAT binding protein (e.g., an antibody), e.g., where HIV infection is used to select a patient.
Exemplary diseases that can be diagnosed using the binding proteins (e.g., antibodies) of the invention include TAT-related diseases, such as diseases characterized by HIV infection, including AIDS.
The methods of the invention may be practiced in conjunction with any other method used by those skilled in the art to provide a diagnosis of the occurrence or recurrence of TAT-related disease. It should be understood that the diagnostic and monitoring methods provided herein are generally used in conjunction with other methods known in the art. For example, the methods of the present invention may be practiced in conjunction with health checks and other known diagnostic methods.
Methods for assessing the efficacy of a treatment regimen in a subject are also provided. In these methods, the amount of TAT peptide in a pair of samples (a first sample obtained from the subject at an earlier time point or prior to a treatment regimen and a second sample obtained from the subject at a later time point (e.g., a later time point when the subject has undergone at least a portion of a treatment regimen) is assessed. It will be appreciated that the methods of the invention include obtaining and analyzing more than two samples (e.g., 3,4, 5,6, 7, 8, 9 or more samples) at regular or irregular intervals to assess TAT peptide levels. Pairwise comparisons can be made between consecutive or non-consecutive subject samples.
The present invention provides methods for monitoring treatment of a TAT-associated disease in a subject by (1) contacting a first biological sample obtained from the subject with a detection reagent specific for TAT peptide prior to administering at least a portion of a treatment regimen to the subject; (2) Contacting a second biological sample obtained from the subject with a detection reagent specific for TAT peptide following administration of at least a portion of the treatment regimen to the subject; (3) Measuring the amount of TAT peptide detected in each of the first and second biological samples by each detection reagent; and (4) comparing the level of expression of the TAT peptide in the first sample with the level of expression of the TAT peptide in the second sample, thereby monitoring treatment of the TAT-related disease in the subject.
In certain embodiments, the diagnostic and monitoring methods provided herein further comprise obtaining a subject sample.
In certain embodiments, the diagnostic and monitoring methods provided herein further comprise selecting a treatment regimen for the subject based on the detected level of TAT peptide.
1. Diagnostic method
Binding proteins (e.g., antibodies or antigen-binding fragments thereof) of the invention are useful in methods of detecting, quantifying, isolating and/or purifying TAT peptides (e.g., TAT protein transduction domains). In some embodiments of the methods provided herein, the TAT protein transduction domain is comprised in a TAT fusion molecule. Thus, in some embodiments, a binding protein (e.g., an antibody or antigen-binding fragment thereof) of the invention can be used in methods of detecting, quantifying, isolating, and/or purifying a TAT fusion molecule. It is to be understood that any of the methods provided herein can be applied to TAT peptides, TAT protein transduction domains, and/or TAT fusion molecules.
Exemplary methods for detecting the presence or absence or amount or level of a TAT peptide (e.g., a TAT protein transduction domain or TAT fusion molecule) in a biological sample include: a biological sample is obtained from a test subject and contacted with a binding protein (e.g., an antibody or antigen-binding fragment thereof) of the invention to detect a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule).
Methods provided herein for detecting the presence or absence or amount or level of a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule) in a biological sample include: obtaining a biological sample from a subject that may or may not contain a TAT peptide to be detected (e.g., a TAT protein transduction domain or a TAT fusion molecule), contacting the sample with a TAT peptide binding protein (e.g., an antibody or antigen-binding fragment thereof) as described herein, and contacting the sample with a detection reagent for detecting the TAT peptide (e.g., a TAT protein transduction domain-specific binding agent complex or a TAT fusion molecule-specific binding agent complex, if formed).
The methods comprise forming a transient or stable complex between a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule) and a TAT peptide antibody or antigen binding fragment thereof described herein. The method requires that the complex, if formed, is formed for a sufficient time to allow the detection reagent to bind to the complex and produce a detectable signal (e.g., a fluorescent signal, a signal from the product of an enzymatic reaction (e.g., a peroxidase reaction, a phosphatase reaction, a beta-galactosidase reaction, or a polymerase reaction)).
Intact antibodies or fragments or derivatives thereof (e.g., fab or F (ab') 2 ) Can be used in the process of the invention. This TAT peptide detection strategy is used, for example, in ELISA, RIA, immunoprecipitation, western blotting, fluorescent imaging of antibody labels, tissue immunohistochemistry, immunoprecipitation or immunopurification followed by mass spectrometry, such as immunoprecipitation-multiplex reaction monitoring (IPMRM) and immunoentangl photometry. In certain embodiments, the TAT peptide-specific binding agent complex is attached to a solid support for detecting a TAT peptide (e.g., a TAT protein transduction domain or TAT fusion molecule), e.g., in an ELISA, RIA, immunoprecipitation, western blot, immunoentanglementry assay. The complex may be formed on the substrate or formed prior to capture on the substrate. For in-gel enzymatic assays, TAT peptides (e.g., TAT protein transduction domains or TAT fusion molecules) are broken down in a gel (typically an acrylamide gel) into which the substrate of the enzyme is incorporated.
In another aspect, the present application provides methods for detecting the presence of a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule) in vivo (e.g., imaging in a subject in vivo). The methods can be used to detect the presence of a TAT peptide (e.g., a TAT protein transduction domain), or to detect or quantify a TAT fusion molecule, or to determine the localization of a TAT fusion molecule or to verify delivery of a TAT fusion molecule in a subject. In an exemplary embodiment, a method comprises: (i) Administering to the subject or a control subject an anti-TAT peptide antibody or fragment thereof as described herein under conditions that allow the antibody or fragment to bind to TAT peptide; and (ii) detecting complex formation between the antibody or fragment and TAT peptide, wherein a statistically significant change in said complex formation in said subject relative to said control subject is indicative of the presence of TAT peptide (e.g., a TAT protein transduction domain or TAT fusion molecule).
In some embodiments, the methods provided herein comprise detecting the presence or absence or amount or level of a TAT peptide (e.g., a TAT protein transduction domain or TAT fusion molecule) in a biological sample obtained from a subject by contacting the biological sample with a TAT peptide binding protein (e.g., an antibody or antigen-binding fragment thereof) of the invention to form a complex between the TAT peptide (e.g., a TAG protein transduction domain or TAT fusion molecule) and the TAT peptide binding protein and purifying the complex. In some embodiments, the TAT peptide binding protein may be immobilized on a solid support (e.g., a plate, bead, or chromatography resin). In further embodiments, the methods provided herein can comprise mass spectrometry-based assays to detect the presence or absence or amount or level of a TAT peptide (e.g., a TAT protein transduction domain or TAT fusion molecule) after it has been purified as part of a complex as described above.
In some embodiments, the methods provided herein comprise administering a TAT fusion molecule to a TAT peptide binding protein (e.g., an antibody or antigen binding fragment thereof) of the invention by contacting a biological sample obtained from a subject with the TAT peptide binding protein to form a complex between the TAT fusion molecule and the TAT peptide binding protein; purifying the complex; and detecting the presence or absence or amount or level of a TAT fusion molecule by mass spectrometry of at least a portion of said TAT fusion molecule. In some embodiments, the TAT peptide binding protein may be immobilized on a solid support (e.g., a plate, bead, or chromatography resin).
In some embodiments, the methods provided herein comprise forming a complex between a TAT fusion molecule and a TAT-peptide binding protein by contacting a biological sample obtained from a subject with a TAT-peptide binding protein of the invention (e.g., an antibody or antigen binding fragment thereof) immobilized on a solid support; purifying the complex; treating the complex with a protease (e.g., trypsin) to produce at least one peptide derived from a TAT fusion molecule; and detecting the presence or absence or amount or level of a TAT fusion molecule by mass spectrometric analysis of the at least one peptide. In some embodiments, the TAT fusion molecule may be a TAT ataxin fusion molecule.
It has been found that TAT peptide binding proteins known in the art, such as the commercially available anti-TAT mouse monoclonal IgM antibody (ab 63957) from Abcam, are characterized by underperforming when used in methods for detecting the presence or absence or amount or level of TAT ataxin fusion molecules in a biological sample. For example, it has been found that when a commercially available anti-TAT antibody is used to immunopurify a TAT ataxin fusion molecule from a biological sample, followed by trypsinization of the immunopurified complex and determination of the level of tryptic peptide by liquid chromatography and mass spectrometry (LC/MS), the resulting mass spectrometry signal is lower than that obtained in a similar experiment using a commercially available anti-TAT antibody for immunopurification. This result indicates that commercially available anti-TAT antibodies are not able to immunopurify sufficient quantities of TAT-ataxin fusion molecules from biological samples to allow reliable detection and quantification of TAT-ataxin fusion molecules. The TAT peptide binding proteins of the invention overcome the above-described underperformance problems and allow for reliable detection and quantification of TAT fusion molecules (e.g., TAT ataxin fusion molecules).
Thus, in some aspects, methods are provided for detecting the presence or quantifying the level of a TAT fusion molecule in a sample comprising contacting the sample with a TAT peptide binding protein (e.g., an antibody or antigen binding fragment thereof) of the invention to form a complex between the TAT fusion molecule and the TAT peptide binding protein, thereby detecting the presence or quantifying the level of the TAT fusion molecule in the sample, wherein the TAT peptide binding protein exhibits a specific binding affinity for the TAT fusion molecule that is greater than the specific binding affinity of an anti-TAT mouse monoclonal IgM antibody from Abcam (ab 63957) for the TAT fusion molecule. In some embodiments, specific binding affinity is measured under conditions comprising one or more of ambient temperature (e.g., 20-25 ℃), a pH of about 7.4, and Phosphate Buffered Saline (PBS) buffer.
In some embodiments, TAT peptide binding proteins of the invention may be characterized by K D K of bia 63957 D Low (i.e., higher affinity) by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 100-fold, or at least about 500-fold. In some embodiments, K D Measured under conditions comprising one or more of ambient temperature (e.g., 20-25 ℃), a pH of about 7.4, and Phosphate Buffered Saline (PBS) buffer.
In some embodiments, the TAT fusion molecule is a TAT ataxin fusion molecule. In some embodiments, the sample can be a biological sample, e.g., a liquid sample such as a blood sample, a plasma sample, or a serum sample, or a solid sample (e.g., a tissue sample such as a skin sample or an oral sample).
In some aspects, methods are provided for isolating or purifying a TAT fusion molecule from a sample, comprising contacting the sample with a TAT peptide binding protein (e.g., an antibody or antigen-binding fragment thereof) of the invention to form a complex between the TAT fusion molecule and the TAT peptide binding protein; and isolating or purifying the complex from the sample; wherein the TAT peptide binding protein exhibits a specific binding affinity for the TAT fusion molecule that is greater than the specific binding affinity of an anti-TAT mouse monoclonal IgM antibody (ab 63957) from Abcam to the TAT fusion molecule. In some embodiments, specific binding is measured under conditions comprising one or more of ambient temperature (e.g., 20-25 ℃), a pH of about 7.4, and Phosphate Buffered Saline (PBS) buffer.
In some embodiments, TAT peptide binding proteins of the invention may be characterized by K D K of bia 63957 D Low (i.e., higher affinity) is at leastAbout 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 100 times, or at least about 500 times. In some embodiments, K D Measured under conditions comprising one or more of ambient temperature (e.g., 20-25 ℃), a pH of about 7.4, and Phosphate Buffered Saline (PBS) buffer.
In some embodiments, the TAT fusion molecule is a TAT ataxin fusion molecule. In some embodiments, the sample may be a biological sample, e.g., a liquid sample such as a blood sample, a plasma sample, or a serum sample, or a solid sample (e.g., a tissue sample such as a skin sample or an oral sample).
In some aspects, there is provided a method for detecting the presence of or quantifying the level of a TAT fusion molecule in a sample, comprising:
(a) Contacting a sample with a TAT peptide binding protein (e.g., an antibody or antigen-binding fragment thereof) of the invention to form a complex between a TAT fusion molecule and the TAT-peptide binding protein,
(b) Purifying the complex;
(c) Treating the complex with a protease (e.g., trypsin) to produce at least one peptide derived from a TAT fusion molecule; and
(d) Analyzing the at least one peptide by mass spectrometry, thereby generating a mass spectrometry signal corresponding to the peptide;
wherein the mass spectral signal generated in step (d) is at least about 2 to about 10 fold higher than the mass spectral signal generated when step (a) is performed using an anti-TAT mouse monoclonal IgM antibody from Abcam (ab 63957), e.g., at least about 3 fold higher, at least about 4 fold higher, at least about 5 fold higher, at least about 6 fold higher, at least about 7 fold higher, at least about 8 fold higher, at least about 9 fold higher, or at least about 10 fold higher.
In some embodiments, step (a) can be performed under conditions comprising one or more of ambient temperature (e.g., 20-25 ℃), a pH of about 7.4, and Phosphate Buffered Saline (PBS) buffer.
In some embodiments, the TAT fusion molecule is a TAT ataxin fusion molecule. In some embodiments, the sample may be a biological sample, which may be a liquid sample (e.g., a blood sample, a plasma sample, or a serum sample) or a solid sample (e.g., a tissue sample, such as a skin sample or an oral sample).
E. Reagent kit
The present invention provides compositions and kits for detecting, quantifying, purifying, and/or isolating TAT peptides, particularly TAT fusion molecules comprising a TAT protein transduction domain. The invention also provides compositions and kits for diagnosing or monitoring TAT-related disease or the recurrence of TAT-related disease.
These kits comprise one or more of the following: a detectable binding protein (e.g., an antibody) that specifically binds to a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain), reagents for obtaining and/or preparing a tissue sample from a subject for staining, and instructions for use. In one embodiment, the binding protein (e.g., antibody) is any one or more of the binding proteins described herein.
The invention also encompasses kits for detecting the presence of a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain) in a biological sample. Such kits can be used to detect, quantify, purify, and/or isolate TAT protein transduction domains or TAT fusion molecules comprising TAT protein transduction domains. Such kits may also be used to determine whether a subject has a TAT-related disease. For example, a kit can comprise a labeled compound or reagent capable of detecting a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain) in a biological sample, and a means for detecting and/or quantifying and/or isolating and/or purifying a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain) in a sample.
The kit can also comprise instructions for use of the kit for practicing any of the methods provided herein or interpreting results obtained using the kit based on the teachings provided herein. The kit may further comprise reagents for detecting a control protein not associated with a TAT-related disease in a sample, e.g., actin in a tissue sample, albumin in a blood or blood-derived sample for normalizing the amount of target antigen present in the sample. The kit can also contain a purified TAT peptide for detection (e.g., a TAT fusion molecule comprising a TAT protein transduction domain) as a control or for quantification of the detection performed with the kit.
Kits comprise reagents for use in methods of diagnosing a TAT-related disease (e.g., HIV infection or AIDS) in a subject, comprising a detection reagent, e.g., an antibody of the invention, wherein the detection reagent is specific for a TAT peptide (e.g., a TAT protein transduction domain or a TAT fusion molecule comprising a TAT protein transduction domain). In one embodiment, the detection reagent is any one or more of the binding proteins described herein.
For antibody-based kits, the kit can comprise, for example: (1) A first antibody (e.g., attached to a solid support) that binds to a first target protein (e.g., a TAT peptide); and optionally, (2) a second, different antibody that binds to the first target protein or the first antibody and is coupled to a detectable label.
Reagents that specifically detect TAT peptides (e.g., TAT fusion molecules comprising a TAT protein transduction domain) allow the detection and quantification of TAT peptides (e.g., TAT fusion molecules comprising a TAT protein transduction domain) in complex mixtures (e.g., serum or tissue samples). In certain embodiments, a kit for detecting, quantifying, isolating or purifying a TAT peptide (e.g., a TAT fusion molecule comprising a TAT protein transduction domain) or for diagnosing or monitoring a TAT-related disease comprises at least one reagent that specifically detects a TAT peptide (e.g., a TAT fusion molecule comprising a TAT protein transduction domain).
In certain embodiments, the kit further comprises instructions for detecting, quantifying, isolating, or purifying a TAT peptide (e.g., a TAT fusion molecule comprising a TAT protein transduction domain) or for diagnosing or monitoring a TAT-related disease based on the level of expression of the TAT peptide (e.g., a TAT fusion molecule comprising a TAT protein transduction domain).
In certain embodiments, the kit may further comprise, but is not limited to, any of buffers, preservatives, protein stabilizers, reaction buffers. The kit may further comprise components necessary for the detection of a detectable label (e.g., an enzyme or substrate). The kit may also contain a control sample or a series of control samples, which may be determined and compared to the test sample. The control can be a control serum sample or a control sample of purified protein or nucleic acid (as the case may be) with known levels of the target TAT peptide. Each component of the kit may be packaged in a separate container, and all of the different containers may be in separate packages with instructions for interpreting the results of the assays performed using the kit.
The kits of the invention may optionally comprise other components for carrying out the methods of the invention.
Pharmaceutical composition
The invention also provides pharmaceutical compositions comprising a binding protein (e.g., an antibody or antigen-binding portion thereof) of the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the antibodies of the invention are useful, but not limited to, the treatment of TAT-related diseases and/or for research purposes. In a specific embodiment, the pharmaceutical composition comprises one or more binding proteins (e.g., an antibody of the invention). According to these embodiments, the composition may further comprise a carrier, diluent or excipient.
The binding proteins (e.g., antibodies or antibody portions) of the invention are incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
Various delivery systems are known and can be used to administer one or more antibodies of the invention, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., wu and Wu, j.biol.chem.262:4429-4432 (1987)), nucleic acid constructs as part of a retrovirus or other vector, and the like. Methods of administering the antibodies of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes).
The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated according to conventional methods into a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic (e.g., lidocaine) to reduce pain at the injection site.
As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired result.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein may be made and may use suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. Having now described the invention in detail, it will be more clearly understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to be limiting of the present invention.
Examples
Example 1: generation of anti-TAT peptide antibodies
Experiments were performed to generate rabbit monoclonal antibodies directed against the TAT protein transduction domain. Rabbit polyclonal and monoclonal antibodies are produced, as well as expression plasmids encoding rabbit immunoglobulins for recombinant production of monoclonal antibodies against TAT moieties (TAT protein transduction domains).
It was found that polyclonal antibodies directed against the entire TAT-FXN fusion molecule recognize the drug, but not in a TAT-specific manner (see FIG. 1). However, polyclonal antibodies raised against KLH-TAT do not recognize mature ataxin, but recognize BSA-TAT, which shows specificity for TAT epitopes (see FIG. 2).
Briefly, rabbits were immunized with TAT, and then sera were collected, affinity purified, and spleen hybridoma fused to prepare rabbit monoclonal antibodies (rabmabs).
Lead optimization included post-fusion hybridoma selection, rabMab expression characterization by EIA screening, limiting dilution well plate inoculation, single cell clone isolation and VH and VL sequencing, followed by production of multiple milligrams of antibody.
Method
Immunization in rabbits
Polyclonal antibodies were obtained by immunizing rabbit E8332 with TAT peptide antigen (MYGRKKRRQRRR-C, SEQ ID NO: 46) coupled to KLH or OVA. The TAT peptide antigen used comprised the sequence YGRKKRRQRRR (SEQ ID NO: 23) of the TAT protein transduction domain. Rabbits received 4 rounds of TAT peptide by subcutaneous injection prior to the first bleed (bleed 1), followed by another injection and a second bleed (bleed 2). After blood 2 was taken, TAT peptide was injected intravenously, and then spleen cells were isolated. Blood 1 and 2 were pooled and purified by affinity purification with TAT peptide antigen to generate polyclonal anti-TAT E8332 polyclonal antibodies. Serum titer data and ELISA showed that E8332 was reactive to both TAT-ataxin fusion molecules and TAT peptide antigen, but not to ataxin alone.
anti-TAT monoclonal antibodies were generated using hybridoma technology. After fusion of spleen cells and myeloma cells, anti-TAT hybridomas are selected and clones are grown in semi-solid HAT selection medium (hypoxanthine-aminopterin-thymidine medium). Clones were grown to sufficient numbers in 24-well multicellular cultures. Single cell seeding in 40 96 well culture plates allowed isolation of clones from a single hybridoma cell per well. Finally, ELISA was performed on the plate-bound BSA-TAT-ataxin fusion protein and BSA-ataxin. Polyclonal screening (subtractive interpretation) against the double antigen allows the identification of clones directed against TAT in the form of a TAT-ataxin fusion molecule (BSA-TAT-ataxin) but not against the sole ataxin (BSA-ataxin).
Cloning of VH and VL sequences from hybridomas
To determine the CDR sequences, total RNA was isolated from hybridoma cells. First and second strand cDNA synthesis was performed. The PCR products were separated by agarose electrophoresis, and the fragments were excised and purified. The fragment was cloned directly into an expression vector. Clones from each reaction were scaled up for miniprep scale plasmid purification.
Identification of functional recombinant VH and VL sequences
For each hybridoma, each plasmid was sequenced. DNA sequencing and analysis were performed on these plasmids.
As a result, the
Polyclonal antibodies
Experiments were performed to compare the ability of rabbit polyclonal antibodies generated as described in the methods section herein to immunopurify exemplary TAT-ataxin fusion molecules with commercially available anti-ataxin (ab 124680, ab113691, ab 110328) and anti-TAT (ab 63957) antibodies. The antibodies used in the experiments are described in the table below. Commercially available antibodies are from Abcam.
Figure BDA0003871364320000791
The 5 antibodies were first biotinylated and then coupled to streptavidin-coated magnetic beads. Antibody-coupled beads were added to plasma samples containing the exemplary TAT ataxin fusion molecule and incubated in PBS at room temperature for binding to occur. After incubation, the beads were washed with PBS and released into digestion buffer. Trypsin was added to produce the trypsin peptide of the exemplary TAT-ataxin fusion molecule. After digestion, formic acid was added to stop digestion, the beads were removed and the digested sample was transferred to a clean culture plate for injection into a liquid chromatography/mass spectrometry (LC/MS) system. In the LC/MS experiment, the intensity of the peak corresponding to the ataxin-derived tryptic peptide GLNQIWNVK (SEQ ID NO: 47) was determined.
The results of the experiment are shown in fig. 3. Specifically, FIG. 3 is a bar graph showing the relative peak areas generated by the ataxin-derived tryptic peptide GLNQIWNVK (SEQ ID NO: 47) following immunopurification of an exemplary TAT ataxin fusion molecule with a test antibody. The results presented in FIG. 3 indicate that the LC/MS signal generated when anti-TAT antibody ab63957 was used for immunopurification was about 5-fold lower than when anti-ataxin antibody ab110328 was used. The results also show that immunopurification using anti-TAT polyclonal rabbit antibody produced about 5-fold higher LC/MS signals than using the commercially available anti-TAT antibody ab63957, and similar to LC/MS signals produced using the anti-ataxin antibody ab 110328. These results demonstrate that the polyclonal rabbit antibodies produced are much more effective in immunopurifying the exemplary TAT ataxin fusion molecule than the commercially available anti-TAT antibody ab 63957. These results indicate that subsequently generated anti-TAT monoclonal antibodies will have at least the same or superior ability to immunopurify TAT fusion molecules as polyclonal antibodies.
Monoclonal antibodies
Monoclonal antibodies directed against TAT protein transduction domains are produced by the hybridoma methods described herein.
The 10 antibodies were converted into recombinant antibodies directed against the TAT protein transduction domain. These antibodies are designated 10-1, 10-4, 10-5, 10-9, 10-12, 12-1, 12-3, 12-8, 12-10 and 6.3. The complete amino acid sequences of the heavy and light chains from these 10 antibodies are shown in Table 1 below as SEQ ID NOS: 1-22.
Antibodies 10-4, 10-5, 12-1, 12-3 have the same heavy and light chain variable region sequences; antibodies 10-9, 12-8, 12-10 have the same heavy and light chain variable region sequences; antibody 10-12 and antibody 10-1 have the same heavy and light chain variable region sequences. Antibody 6.3 has unique heavy and light chain variable region sequences.
All antibodies, except one (antibody 6.3), had the same heavy chain CDR sequences. Thus, high affinity anti-TAT monoclonal antibodies exist as a common set of heavy chain CDRs (SEQ ID NOS: 2, 3 and 4).
Antibodies 10-1, 10-9, 10-12, 12-8, and 12-10 have identical light chain CDR sequences, and antibodies 10-4, 10-5, 12-1, and 12-3 have identical light chain CDR sequences. Thus, high affinity anti-TAT monoclonal antibodies exist as two sets of light chain CDRs in common (SEQ ID NOS: 6,7 and 8 and SEQ ID NOS: 10, 11 and 12).
Table 1: anti-TAT human antibody heavy and light chain variable region amino acid sequences
Figure BDA0003871364320000811
Figure BDA0003871364320000821
Figure BDA0003871364320000831
Figure BDA0003871364320000841
Figure BDA0003871364320000851
The nucleic acid sequences of the heavy and light chain variable regions of the 10 antibodies are shown in table 2 below.
Table 2: anti-TAT human antibody heavy and light chain variable region nucleic acid sequences
Figure BDA0003871364320000852
Figure BDA0003871364320000861
Figure BDA0003871364320000871
Figure BDA0003871364320000881
Figure BDA0003871364320000891
Figure BDA0003871364320000901
Figure BDA0003871364320000911
Figure BDA0003871364320000921
Example 2 screening for clones that specifically bind to TAT-ataxin fusion molecules
Sequences of heavy and light variable regions of the 10 high affinity monoclonal clones described in example 1 were cloned into different rabbit IgG backbone sequences to generate test plasmids. Sandwich ELISA was modified by using polyclonal anti-TAT capture to screen for clones with appropriate capture sensitivity to TAT-containing proteins as follows: ELISA plates were coated with 50. Mu.L of 5. Mu.g/ml purified expressed antibody from clonal cells transfected with the test plasmid and then blocked with high protein buffer. A calibration solution of 50. Mu.L buffer supplemented with TAT-ataxin at a concentration ranging from 1000ng/ml to 0.05ng/ml was incubated in the coated wells for 2 hours at room temperature and then washed. Then, containing HRP labeled known anti-ataxin monoclonal antibody 50 u L buffer in the hole were incubated for 1 hours, followed by washing, and with 100L TMB substrate incubated for 20 minutes. The detection reaction was stopped by adding 100. Mu.L of 1N H2SO4 and the plate was read at 450 nm. Softmax was used to fit curves to determine sensitivity and compare antibodies expressing the clones.
In a Pharmacokinetic (PK) assay in human plasma, 9 of the 10 antibodies identified (except antibody 6.3) were able to effectively capture an exemplary TAT fusion molecule comprising a TAT protein transduction domain and an ataxin (see fig. 4A and 4B).
Antibodies were tested for specific binding to exemplary TAT FXN fusion molecules in a binding assay in human plasma. The binding assay used is an ADA or bridging assay, generally as described in example 3 below. The results of the binding assay are shown in fig. 5A. The results show that all recombinant antibodies tested, except one antibody (11563-1), showed specific binding to the TAT FXN fusion molecule. The results also show that the sequences of the heavy and light chain variable regions of the anti-TAT monoclonal antibody confer the ability to specifically bind to the TAT-FXN fusion molecule when placed in different backbones. This confirms the discovery of high affinity rabbit antibodies. All clones except clone 11563-1 showed a hook effect at high antibody concentrations, as expected from the bridge/sandwich assay, where capture was dependent on a single defined epitope (TAT).
Example 3 anti-TAT polyclonal antibodies are effective as a positive control in ADA assays
The purpose of this experiment was to compare rabbit polyclonal anti-TAT antibodies generated by the methods described herein with commercially available anti-ataxin antibodies to determine whether the rabbit polyclonal anti-TAT antibodies can be used in anti-drug antibody (ADA) assays to test the immunogenicity of exemplary TAT ataxin fusion molecules.
In the assay, two labeled forms (biotinylated and sulfonated labeled forms) of the exemplary TAT ataxin fusion molecule were incubated with antibodies having defined concentrations. The samples were incubated on streptavidin plates, unbound molecules washed away, and the plates were then read to see the amount of labeled (sulfonate-labeled) antibody binding. This is a bridging strategy, in which a high affinity molecule capable of binding two TAT ataxin molecules completes the "bridging", resulting in signal binding to the plate.
The results are shown in fig. 5B. Specifically, the results demonstrate that commercial anti-ataxin monoclonal antibodies exhibit a strong bridging signal up to about 10 μ g/ml antibody input, and a reduced signal at higher concentrations, which is commonly described as a "hook effect". The rabbit polyclonal anti-TAT antibody produced a signal that increased up to about 50. Mu.g/ml, and maintained a maximum signal to a concentration of about 200. Mu.g/ml. The results indicate that polyclonal anti-TAT antibodies can function well as a positive control in ADA assays to screen clinical samples for potential antibodies generated, for example, by treatment with TAT-ataxin fusion molecules.
Sequence listing
Figure BDA0003871364320000941
Figure BDA0003871364320000951
Figure BDA0003871364320000961
Equivalents of
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the appended claims.
Figure IDA0003871364360000011
Figure IDA0003871364360000021
Figure IDA0003871364360000031
Figure IDA0003871364360000041
Figure IDA0003871364360000051
Figure IDA0003871364360000061
Figure IDA0003871364360000071
Figure IDA0003871364360000081
Figure IDA0003871364360000091
Figure IDA0003871364360000101
Figure IDA0003871364360000111
Figure IDA0003871364360000121
Figure IDA0003871364360000131
Figure IDA0003871364360000141
Figure IDA0003871364360000151
Figure IDA0003871364360000161
Figure IDA0003871364360000171
Figure IDA0003871364360000181
Figure IDA0003871364360000191
Figure IDA0003871364360000201
Figure IDA0003871364360000211
Figure IDA0003871364360000221
Figure IDA0003871364360000231
Figure IDA0003871364360000241
Figure IDA0003871364360000251
Figure IDA0003871364360000261

Claims (83)

1. A binding protein comprising an antigen binding domain comprising a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID No. 4, wherein the binding protein is capable of binding to a TAT protein transduction domain.
2. The binding protein according to claim 1, wherein said antigen binding domain further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3.
3. The binding protein according to claim 1 or 2, wherein said antigen binding domain further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2.
4. The binding protein according to any one of claims 1-3, wherein said antigen binding domain further comprises a light chain CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 8 and SEQ ID NO 12.
5. The binding protein according to any one of claims 1-4, wherein said antigen binding domain further comprises a light chain CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 7 and SEQ ID NO 11.
6. The binding protein according to any one of claims 1-5, wherein said antigen binding domain further comprises a light chain CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 6 and SEQ ID NO 10.
7. A binding protein comprising an antigen binding domain, the antigen binding domain comprising:
a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 4, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 3, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 2; and
a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 8, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 7, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 6; or a light chain variable region comprising a CDR3 domain comprising the amino acid sequence shown in SEQ ID NO. 12, a CDR2 domain comprising the amino acid sequence shown in SEQ ID NO. 11 and a CDR1 domain comprising the amino acid sequence shown in SEQ ID NO. 10,
wherein the binding protein is capable of binding a TAT protein transduction domain.
8. The binding protein according to any one of claims 1-7, wherein said antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 1 or SEQ ID NO 14.
9. The binding protein according to any one of claims 1-8, wherein said antigen binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 5, a light chain variable region comprising the amino acid sequence set forth in SEQ ID No.9, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 13.
10. The binding protein according to any one of claims 1-9, wherein said antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 5.
11. The binding protein according to any one of claims 1-9, wherein said antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 9.
12. The binding protein according to any one of claims 1-9, wherein said antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 13.
13. The binding protein according to any one of claims 1-9, wherein said antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 14 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 5.
14. A binding protein comprising an antigen binding domain comprising a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID No. 18, wherein the binding protein is capable of binding to a TAT protein transduction domain.
15. The binding protein according to claim 14, wherein said antigen binding domain further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID No. 17.
16. The binding protein according to claim 14 or 15, wherein said antigen binding domain further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID No. 16.
17. The binding protein according to any one of claims 14-16, wherein said antigen binding domain further comprises a light chain CDR3 domain comprising the amino acid sequence of SEQ ID No. 22.
18. The binding protein according to any one of claims 14-17, wherein said antigen binding domain further comprises a light chain CDR2 domain comprising the amino acid sequence of SEQ ID No. 21.
19. The binding protein according to any one of claims 14-18, wherein said antigen binding domain further comprises a light chain CDR1 domain comprising the amino acid sequence of SEQ ID No. 20.
20. A binding protein comprising an antigen binding domain, the antigen binding domain comprising:
a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 18, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 17, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 16; and
a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO. 22, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO. 21, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO. 20,
wherein the binding protein is capable of binding a TAT protein transduction domain.
21. The binding protein according to any one of claims 14-20, wherein said antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 15.
22. The binding protein according to any one of claims 14-21, wherein said antigen binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 19.
23. The binding protein according to any one of claims 14-22, wherein said antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 15 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 19.
24. The binding protein according to any one of claims 1-23, wherein the TAT protein transduction domain comprises the amino acid sequence of SEQ ID No. 23.
25. The binding protein according to any one of claims 1-23, wherein the TAT protein transduction domain consists essentially of the amino acid sequence of SEQ ID No. 23.
26. The binding protein according to any one of claims 1-25, wherein the TAT protein transduction domain is covalently linked to a cargo moiety.
27. The binding protein according to claim 26, wherein the cargo moiety is a polypeptide.
28. The binding protein according to claim 26, wherein the cargo moiety is an ataxin polypeptide.
29. The binding protein according to claim 26, wherein the cargo moiety is an antibody.
30. The binding protein according to claim 26, wherein the cargo moiety is a nucleic acid.
31. The binding protein according to claim 30, wherein the nucleic acid is an siRNA, shRNA, miRNA, phosphorothioate modified RNA, aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof.
32. The binding protein according to claim 26, wherein said cargo moiety is a small molecule, a liposome-encapsulated protein, a radionuclide or a radionuclide-labeled compound, or any combination thereof.
33. A binding protein comprising an antigen binding domain, wherein the binding protein is capable of binding to a TAT protein transduction domain covalently linked to a cargo moiety.
34. The binding protein according to claim 33, wherein said antigen binding domain binds to an epitope comprising amino acid residues of SEQ ID No. 23.
35. The binding protein according to claim 33, wherein the cargo moiety is a polypeptide.
36. The binding protein according to claim 33, wherein the cargo moiety is an ataxin polypeptide.
37. The binding protein according to claim 33, wherein the cargo moiety is an antibody.
38. The binding protein according to claim 33, wherein the cargo moiety is a nucleic acid.
39. The binding protein according to claim 38, wherein the nucleic acid is an siRNA, shRNA, miRNA, phosphorothioate modified RNA, aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof.
40. The binding protein according to claim 33, wherein said cargo moiety is a small molecule, a liposome-encapsulated protein, a radionuclide or a radionuclide-labeled compound, or any combination thereof.
41. The binding protein according to any one of claims 1-40, wherein said binding protein is an antibody.
42. An antibody construct comprising the binding protein of any one of claims 1-41, said antibody construct further comprising a linker polypeptide or an immunoglobulin constant region.
43. The antibody construct according to claim 42, wherein the binding protein is selected from the group consisting of: immunoglobulin molecules, monoclonal antibodies, murine antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, single domain antibodies, fv, disulfide-linked Fv, scFv, diabody, fab ', F (ab') 2 Multispecific antibodies, dual specific antibodies and bispecific antibodies.
44. Any one of claims 42 and 43The antibody construct of (a), wherein the binding protein comprises a heavy chain immunoglobulin constant region selected from the group consisting of: igM constant region, igG 4 Constant region, igG 1 Constant region, igE constant region, igG 2 Constant region, igG 3 Constant region and IgA constant region.
45. The antibody construct of claim 44, wherein the binding protein comprises IgG 1 A constant region.
46. The antibody construct of claim 44, wherein the heavy chain immunoglobulin constant region is not IgM.
47. An isolated nucleic acid encoding the binding protein amino acid sequence of any one of claims 1-41.
48. An isolated nucleic acid encoding the antibody construct amino acid sequence of any one of claims 42-46.
49. A vector comprising the isolated nucleic acid of claim 47 or 48.
50. The vector according to claim 49, wherein the vector is selected from the group consisting of: pcDNA, pTT3, pEFBOS, pBV, pJV, pBJ, pGEX, VSV, pBR322, pCMV-HA, pEN, YAC, BAC, lambda phage, phagemid, pCAS9, pCEN6, pYES1L, p3HPRT1, pFN2A, pBC, pTZ, pGEM, pGEMK, pEX, pSAR, pCEP, cosmid, pBluescript, pKJK, pFloxin, pCP, pHR, pUC and AL.
51. A host cell comprising the vector of claim 49 or 50.
52. The host cell of claim 51, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
53. The host cell of claim 51, wherein the prokaryotic host cell is an E.
54. The host cell of claim 51, wherein the eukaryotic cell is selected from the group consisting of: protist cells, insect cells, animal cells, plant cells, and fungal cells.
55. The host cell of claim 51, wherein the animal cell is a mammalian cell or an avian cell.
56. The host cell of claim 51, wherein the host cell is selected from the group consisting of: CHO cells, COS cells, yeast cells, insect Sf9 cells, HEK-293 cells, expiCHO cells, expi-293f cells, and E.coli cells.
57. The host cell of claim 56, wherein the yeast cell is a Saccharomyces cerevisiae cell.
58. A method of producing an antibody, or antigen-binding portion thereof, comprising culturing the host cell of any one of claims 51-57 in a culture medium, thereby expressing the isolated nucleic acid and producing the antibody.
59. An antibody produced according to the method of claim 58.
60. A transgenic mouse comprising the host cell of any one of claims 51-57, wherein the mouse expresses a polypeptide encoded by the nucleic acid, or an antigen-binding portion thereof, that binds to a TAT protein transduction domain.
61. A hybridoma that produces the antibody construct of any one of claims 42-46.
62. The binding protein according to any one of claims 1-41, which is immobilized on a solid support.
63. The binding protein according to claim 62, wherein said solid support is a plate, a bead or a chromatography resin.
64. The binding protein according to claim 63, wherein said bead or chromatography resin comprises protein A Sepharose or protein G Sepharose.
65. The binding protein according to any one of claims 1-41, which is coupled to a detection molecule.
66. The binding protein according to claim 65, wherein the detection molecule is horseradish peroxidase, ruthenium terpyridyl, alkaline phosphatase, cresyl violet, quantum dots, FITC, an infrared molecule, a radioisotope label or an EPR spin tracer tag.
67. A method of detecting and/or quantifying the level of a TAT fusion molecule in a sample, comprising contacting the sample with a TAT protein transduction domain in a sample under conditions such that the binding protein according to any one of claims 1-41 binds to the binding protein, thereby detecting and/or quantifying the level of the TAT fusion molecule in the sample.
68. The method of claim 67, wherein the sample is a biological sample.
69. The method of claim 68, wherein the biological sample is a liquid sample or a tissue sample.
70. The method of claim 68, wherein the TAT fusion molecule comprises a TAT protein transduction domain covalently linked to a cargo moiety.
71. The method of claim 70, wherein the cargo moiety is a polypeptide.
72. The method of claim 70, wherein the cargo moiety is an ataxin polypeptide.
73. The method of claim 70, wherein the cargo moiety is an antibody.
74. The method of claim 70, wherein the cargo moiety is a nucleic acid.
75. The binding protein according to claim 74, wherein the nucleic acid is an siRNA, shRNA, miRNA, phosphorothioate modified RNA, aptamer, phosphorodiamidate Morpholino Oligonucleotide (PMO), or any combination thereof.
76. The binding protein according to claim 70, wherein said cargo moiety is a small molecule, a liposome-encapsulated protein, a radionuclide or a radionuclide-labeled compound, or any combination thereof.
77. The method of claim 67, further comprising assessing the stability of the TAT fusion molecule.
78. A method of isolating and/or purifying a TAT fusion molecule present in a mixture, wherein the TAT fusion molecule comprises a TAT protein transduction domain covalently linked to a cargo moiety, the method comprising (a) contacting the mixture comprising the TAT fusion molecule with an immobilized binding protein of any one of claims 62-64 under conditions such that the TAT fusion molecule binds to the immobilized binding protein; (b) Eluting the TAT fusion molecule from the immobilized binding protein.
79. A kit comprising at least one reagent for specifically detecting TAT protein transduction domain levels, wherein the detection reagent is a binding protein according to any of claims 1-41.
80. The kit of claim 79, wherein the TAT protein transduction domain is covalently linked to a cargo moiety.
81. The kit of claim 79 or 80, wherein the kit further comprises instructions for detecting, quantifying, or characterizing the TAT protein transduction domain.
82. A method of inhibiting translocation of a TAT fusion molecule across a cell membrane, comprising contacting the TAT fusion molecule with an antigen binding protein of any of claims 1-41, thereby inhibiting translocation of the TAT fusion molecule across a cell membrane.
83. A method of inhibiting the activity of HIV-TAT protein in a subject, comprising administering to the subject an antigen-binding protein of any of claims 1-41, thereby inhibiting the activity of HIV-TAT protein in the subject.
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